Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof

ABSTRACT

Cloning, expression, viral and delivery vectors and hosts which contain nucleic acid coding for at least one peripheral nervous system specific (PNS) sodium channel peptide (SCP), isolated PNS SCP, and compounds and compositions and methods, are provided, for isolating, crystallizing, x-ray analysing molecular modeling, rational drug designing, selecting, making and using therapeutic or diagnostic agents or ligands having at least one peripheral nervous system specific (PNS) sodium channel (SC) modulating activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. application.Ser. No. 08/482,401, filed Jun. 7, 1995, which is a continuation-in-partof U.S. application Ser. No. 08/334,029 filed Nov. 2, 1994,both of whichdisclosures are entirely incorporated herein by reference

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

[0002] The present invention was made with U.S. government supportTherefore, the U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is in the fields of biotechnology, proteinpurification and crystallization, x-ray diffraction analysis,three-dimensional computer molecular modeling, and rational drug design(RDD). The invention is directed to isolated peripheral nervous system(PNS) specific sodium channel proteins (SCPs) and encoding nucleic acid,as well as to compounds, compositions and methods for selecting, makingand using therapeutic or diagnostic agents having sodium channelmodulating activity. The present invention further providesthree-dimensional computer modeling of the PNS SCP, and for RDD, basedon the use of x-ray data and/or amino acid sequence data on computerreadable media.

BACKGROUND OF THE INVENTION

[0004] Voltage-sensitive ion channels are a class of transmembraneproteins that provide a basis for cellular excitability, as the abilityto transmit information via ion-generated membrane potentials. Inresponse to changes in membrane potentials, these molecules mediaterapid ion flux through highly selective pores in a nerve cell membrane.If the channel density is high enough, a suitable regenerativedepolarization results, termed the action potential.

[0005] The voltage-sensitive sodium channel is the ion channel mostoften responsible for generating the action potential in excitablecells. Although sodium-based action potentials in different excitabletissues look similar (Hille, B., In: Ionic Channels of ExcitableMembranes, B. Hille, ed., Sinauer, Sunderland, Mass., (1984), pp. 70-71)recent electophysiological studies indicate that sodium channels indifferent cells differ in both their structural and functionalproperties, and many sodium channels with distinct primary structureshave now been identified. See, e.g., Mandel, J. Membrane Biol. 125:193-205 (1992).

[0006] Functionally distinct sodium channels have been described in avariety of neuronal cell types (Llinas et al., J. Physiol. 305:197-213(1980); Kostytk et al., Neuroscience 6:2423-2430(1981); Bossu et al.,Neurosci. Lett. 51:241-246 (1984) 1981, Gilly et al., Nature 309:448-450(1984); French et al, Neurosci. Lett. 56:289-294 (1985); Ikeda et al.,J. Neurophysiol. 55:527-539 (1986); Jones et al., J. Physiol.389:605-627 (1987); Alonso & Llinas, 1989; Gilly et al., J. Neurosci9:1362-1374 (1989)) and in skeletal muscle (Gonoi et al., J. Neurosci5:2559-2564 (1985); Weiss et al., Science 233:361-364 (1986)). Thekinetics of sodium currents in glia and neurons can also bedistinguished (Barres et al., Neuron 2:1375-1388 (1989)).

[0007] The type II and type III genes, expressed widely in the centralnervous system (CNS), are expressed at very low levels in some cells inthe PNS (Beckh, S., FEBS Lett. 262:317-322 (1990)). The type II and IIImRNAs were barely detectable, by Northern blot analysis, in dorsal rootganglion (DRG), cranial nerves and sciatic nerves. On the other hand,type I mRNA was present in moderately high amounts in DRG and cranialnerve, but in low levels in sciatic nerve. A comparison of the amount ofall three brain mRNAs, relative to total sodium channel mRNA detectedwith a conserved cDNA probe, suggested the presence of additional, asyet unidentified, sodium channel types in DRG neurons. Consistent withthe mRNA studies, immunochemical studies showed that neither type I nortype II sodium channel alpha subunits made up a significant component ofthe total sodium channels in the superior cervical ganglion or sciaticnerve (Gordon et al., Proc. Natl. Acad. Sci. USA 84:8682-8686 (1987)).

[0008] A population of neurons in vertebrate DRG has been identifiedelectrophysiologically that contains, in addition to the moreconventional channels, a distinct sodium channel type; this DRG channelhas a k_(D) for TTX approximately tenfold higher than the k_(D) ofsodium channels in either skeletal muscle or heart (Jones et al., J.Physiol. 389:605-627 (1987)).

[0009] The localization of different sodium channels to specific regionsin the nervous system supports the possibility that cell-specificregulation of this gene family is at the transcriptional level. Byanalogy with other eukaryotic genes, distinct DNA elements can bepresent which mediate cell-specific and temporal regulation ofindividual sodium channel genes.

[0010] Studies of sodium channel gene regulation have been facilitatedby the use of well-characterized cell lines, such as pheochromocytoma(PC12) cells, a popular cell model for neuronal differentiation (Greenet al., Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976); Halegoua et al.,Curr. Top. Microbiol. Immunol. 165:119-170 (1991)). In addition toextending neurites and initiating synthesis of certainneurotransmitters, NGF-treated PC12 cells acquire the ability togenerate sodium-based action potentials (Dichter et al., Nature268:501-504 (1977)). This ability is conferred by an increase in thedensity of functional sodium channels in the membranes of theNGF-treated cells (Rudy et al., J. Neurosci. 7:1613-1625 (1987); Mandelet al., Proc. Natl. Acad Sci. USA 85:924-928 (1988); O'Lague et al.,Proc. Natl. Acad. Sci. USA 77:1701-1705 (1980)). Northern blot analysisrevealed that undifferentiated PC12 cells contained a basal level ofsodium channel mRNA which increased coincident with the increase inchannel activity observed after treatment with NGF (Mandel et al., Proc.Natl. Acad. Sci. USA 85:924-928 (1988)).

[0011] There is a long standing need to diagnose and/or treatpathologies relating to impaired peripheral nervous system (PNS) nerveconduction associated with PNS injury or in genetic or other diseasestates, such as those involving lack of or defects in, PNS sodiumchannels (SCs). In view of the possibility of cell or tissue specificsodium channels, the discovery and use of isolated PNS SCs and encodingnucleic acid would provide an opportunity to diagnose or treat suchpathologies by either screening suitable PNS SC modulating drugs ormolecules (e.g., analgesics), or by using recombinant PNS SCs for insitu or in vivo gene therapy to replace or supplement PNS SCs in atleast one portion of the peripheral nervous system of a mammalianpatient suffering from a PNS SC related pathology.

SUMMARY OF THE INVENTION

[0012] The present invention (hereinafter, “invention”) providesperipheral nervous system specific (PNS) sodium channel peptides (SCPs),encoding nucleic acid, vectors, host cells and antibodies, as well asmethods of making and using thereof, including recombinant expression,purification, cell-based drug screening, gene therapy, crystallization,X-ray diffraction analysis, as well as computer structure determinationand rational drug design utilizing at least one PNS SCP amino acidsequence and/or x-ray diffraction data provided on computer readablemedia.

[0013] The invention also includes oligonucleotide probes specific forPNS SCP encoding sequences, as well as methods for dectection in asample, where the probe is labeled. The invention further includesmethods for producing a PNS SCP, comprising culturing a host in aculture medium, comprising a PNS SCP nucleic acid; and isolating the PNSSCP from said host or said culture medium.

[0014] The invention additionally includes an antibody which binds anepitope specific for a PNS SCP, as well as host cells which express theantibody. Diagnostic or therapeutic methods using the antibody are alsoincluded in the invention.

[0015] The invention further includes gene therapy methods and deliveryvectors comprising nucleic acid encoding, or complementary to, at leastone PNS SCP, and pharmaceutically acceptable compositions thereof.

[0016] The invention also includes gene therapy by methods thatadminister an antisense PNS SCP nucleic acid to an animal in amounteffective to provide a PNS SC modulating effect, such as an analgesiceffect.

[0017] The present invention further provides methods for purifying andcrystallizing a PNS SCP that can be analyzed to obtain x-ray diffractionpatterns of sufficiently high resolution to be useful forthree-dimensional molecular modeling of the protein. The x-raydiffraction data, atomic coordinates, and/or amino acid sequencesprovided on computer readable medium, are modeled on computer systems,using methods of the invention, to generate secondary, tertiary and/orquaternary structures of a PNS SCP, which structures contribute to theiroverall three dimensional structure, as well as binding and active sitesof the PNS SCP.

[0018] Molecular modeling methods and computer systems are also providedby the present invention for rational drug design (RDD). These drugdesign methods use computer modeling programs to find potential ligandsor agents that are calculated to bind with sites or domains on the PNSSCP. Potential ligands or agents are then screened for modulating orbinding activity. Such screening methods can be selected from assays forat least one biological activity of the protein, as associated with aPNS SCP-related pathology or trauma, according to known sodium channelassays. The resulting ligands provided by methods of the presentinvention are synthesized and are useful for treating, inhibiting orpreventing at least one of PCS SCP-related pathology or trauma in amammal.

[0019] Further objects, features, utilities, embodiments and/oradvantages of the present invention will be apparent from the additionaldescription provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 depicts a 323 amino acid and corresponding 969 nucleotidesequence of a PNS SCP as amino acids 233-555 of SEQ ID NO:2 andnucleotides 699-1665 of SEQ ID NO:1, as the primary structure of DomainIII of the Peripheral Nerve type I (PN1) sodium channel alpha ( )subunit for both amino acid and DNA sequences. The single amino acidcode is used to denote deduced amino acids. YJ1 and YOIC refer to theoligonucleotide primers used to obtain the initial PCR fragment of PN1cDNA.

[0021]FIG. 2A-B shows a Northern blot analysis of sodium channel αsubunit mRNA in rat pheochromocytoma (PC12) cells treated with NerveGrowth Factor. In FIG. 2(A), the probe used is pRB211 which encodes thehighly conserved fourth repeated domain of the rat type II sodiumchannel. Both type H and PN1 mRNAs are detected with this probe. In FIG.2(B), the probe used contains sequences specific for PN1. The levels ofsodium channel mRNA are quantitated with reference to the amount ofcyclophilin mRNA, as indicated. Control cells are PC 12 cells grown inthe absence of NGF.

[0022]FIG. 3A-B shows an example of tissue-specific distribution of PN1mRNA. FIG. 3(A) presents a Northern blot analysis using equal amounts ofRNA from tissues. PN1 mRNA is indicated by the dash. 28S refers to the28S rRNA. The probe contains sequences specific for the PN1 gene. Notethe absence of PN1 mRNA in skeletal muscle, cardiac muscle, and the lowlevels of PN1 mRNA in spinal cord. FIG. 3(B) shows RNAase protectionanalysis of PN1 mRNA. PN1 refers to the PN1 probe protected by mRNA fromthe different tissue samples. Actin refers to actin probe sequencesprotected by the same mRNA.

[0023]FIG. 4A-F shows localization of PN1 mRNA in Superior CervicalGanglion (SCG) and Dorsal Root Ganglion (DRG) tissues by in situhybridization analysis. FIGS. 4A-4B represent neurons hybridized with aPN1-specific antisense RNA probe. FIGS. 4C-4D represent neuronshybridized with the radiolabeled PN1 probe in the presence ofnon-labeled PN1 competitor DNA. FIGS. 4E-4F represent tissue sectionshybridized with an antisense type II probe.

[0024]FIG. 5 shows a blot analysis comparing Levels of PN1 and braintype I a subunit mRNA in SCG. The pRB11 conserved sodium channel probedetects both type II/IIA and PN1 transcripts.

[0025]FIG. 6A-B shows a Northern blot analysis which revealsdifferential expression of PN1 and type I sodium channel mRNAs duringpostnatal rat development. FIG. 6(A) shows a representativeautoradiogram of a Northern blot using radiolabeled antisense pRB211 RNAas probe. Postnatal days 7 (P7) to 42 (P42) are shown. FIG. 6(B) shows aplot of quantitation of the Northern blots showing a decrease in type ImRNA with time after birth.

[0026]FIG. 7A-D show the deduced primary structure of cloned portion ofPN1 a subunit cDNA as a partial 3033 nucleotide (SEQ ID NO:1) sequenceand a partial 1011 amino acid (SEQ ID NO:2) sequence.

[0027]FIG. 8A-D show a comparison of deduced primary amino acidsequences of PN1 (1-988 of SEQ ID NO:2) and brain type II/IIA α subunit(SEQ ID NO:).

[0028]FIG. 9A-9D show the entire DNA sequence for a rat PN1 PNS SCP(SEQID NO:9).

[0029]FIG. 10 shows the entire amino sequence for a rat PN1 PNS SCP (SEQID NO:10).

[0030]FIG. 11A-11E shows amino acid sequences for rat PN1 (“RATPN1”)(SEQ ID NO:10) and two expected human PN1 sequences “HULMN1A” (SEQ IDNO:11) “HUMPN1B”(SEQ ID NO:12) HUMPN1C (SEQ ID NO:7) and HUMPN1D (SEQ IDNO:). Alternative sequences include those where “X” is 0, 1, 2, or 3 ofthe same or different amino acids, which can be optionally selected fromTable 1 or Table 2.

[0031]FIG. 12 shows a computer system suitable for three dimensionalstructure determination and/or rational drug, design.

[0032]FIG. 13A-B shows a representative DNA sequence encoding a humanPN1 (HUM PN1A) (SEQ ID NO:11)

[0033]FIG. 14B shows a representative DNA sequence encoding a human PN1(HUM PN1B) (SEQ ID NO:12)

DETAILED DESCRIPTION OF THE INVENTION

[0034] A need exists for modulating the activity of at least oneperipheral nervous system specific (PNS) sodium channel (SCs). Suchmodulation could potentially provide analgesic or diagnostic agents forpain or pathologies associated with nerve conduction in the PNS.

[0035] Certain sodium channels—corresponding to PNS SCPs of theinvention—are now discovered to be preferentially or selectivelyexpressed in the peripheral nervous system (PNS). These sodium channelsmodulate peripheral nerve impulse conduction preferentially in the PNS.The present invention provides peripheral nervous system specific (PNS)sodium channel peptides (SCPs), encoding nucleic acid, vectors, hostcells and antibodies, as well as methods of making and using thereof,including recombinant expression, purification, cell-based drugscreening, gene therapy, crystallization, X-ray diffraction analysis, aswell as computer structure determination and rational drug designutilizing at least one PNS SCP amino acid sequence and/or x-raydiffraction data provided on computer readable media.

[0036] A PNS sodium channel peptide (PNS SCP) can refer to any subset ofa PNS sodium channel (SC) having SC activity, as a fragment, consensussequence or repeating unit. A PNS SCP of the invention can be preparedby:

[0037] (a) recombinant DNA methods;

[0038] (b) proteolytic digestion of the intact molecule or a fragmentthereof;

[0039] (c) chemical peptide synthesis methods well-known in the art;and/or

[0040] (d) by any other method capable of producing a PNS SCP and havinga conformation similar to an active portion of a PNS SCP and having SCactivity. The SC activity can be screened according to known screeningassays for sodium channel activity, in vitro, in situ or in vivo. Theminimum peptide sequence to have activity is based on the smallest unitcontaining or comprising a particular region, domain, consensussequence, or repeating unit thereof, of at least one PNS SCP.

[0041] According to the invention, a PNS SCP includes an association oftwo or more polypeptide domains, such as transmembrane, pore liningdomains, or fragments thereof, corresponding to a PNS SCP, such as 1-40domains or any range or value therein. Transmembrane, cytoplasmic porelining or other domains of a PNS SCP of the invention may have at least74% homology, such as 74-100% overall homology or identity, or any rangeor value therein to one or more corresponding SC domains as describedherein (eg., as presented FIGS. 1, 7, 8, 10 or 11). As would beunderstood by one of ordinary skill in the art, the above configurationof domains are provided as part of a PNS SCP of the invention, such thata functional PNS SCP, when expressed in a suitable cell, is capable oftransporting sodium ions across a lipid bilayer, a cell membrane or amembrane model. In intact cells having sufficient sodium channels, thecell can be capable of generating some form of an action potential, suchas in a cell expressing at least one PNS SCP of the present invention.Such transport, as measured by suitable SC activity assays, establishesSC activity of one or more PNS SCPs of the invention.

[0042] Accordingly, a PNS SCP of the invention alternatively includespeptides having a portion of a SC amino acid sequence whichsubstantially corresponds to at least one 20 to 2005 amino acid fragmentand/or consensus sequence of a PNS SCP or group of PNS SCPs, wherein thePNS SCP has homology or identity of at least 74-99%, such as 88-99% (orany range or value therein, e.g., 87-99, 88-99, 89-99, 90-99, 91-99,92-99, 93-99, 94-99, 95-99, 96-99, 97-99, or 98-99%) homology to atleast one sequence or consensis sequence of FIGS. 1, 7, 8, 10 r 11. Inone aspect, such a PNS SCP can maintain SC biological activity. It ispreferred that a PNS SCP of the invention is not naturally occurring oris naturally occurring but is in a purified or isolated form which doesnot occur in nature. Preferably, a PNS SCP of the inventionsubstantially corresponds to an set of domains of PN1, having at least10 contiguous amino acids of FIGS. 1, 7, 8, 10 and 11, or at least 74%homology thereto.

[0043] Alternatively or additionally, a PNS SCP of the invention maycomprise at least one domain corresponding to known sodium channeldomains, such as rat brain or spinal cord SC domains, such astransmembrane domains, pore lining domains, cytoplasmic domains orextracellular domains, such as IIs6 (e.g., 1-3 to 14-17 (IIs6) 18-23 to210-214 (cytoplasmic), 229-236 to 254-258 (IIIS1), 268-272 to 293-297(IIIs2), 300-304 to 321-325 (IIIs3), 326-330 to 347-351 (IIIs4), 368-374to 389-393 (IIIs5), 474-478 to 500-504 (IIIs6), 553-559 to 577-583(IVs1), 589-593 to 611-615 (IVs2), 619-623 to 642-646 (IVs3), 654-658 to678-682 (IVs4), 690-694 to 711-715 (IVs5), 779-783 to 801-805 (IVs6),348-352 to 368-372, 501-505 to 550-554, 233-555, 676-678 to 689-693,554-557 to 941-945, or any range or value therein, corresponding to SEQID NO:2 as presented in FIG. 7A-7D, or variants thereof as presentedsubstitutions in Table 1 or Table 2, having 74-100% overall homology orany range or value therein. At least one of such domains are present inthe PNS SCPs presented in FIG. 11A-E, or fragments thereof, asnon-limiting examples. Alternative domains are also encoded by DNA whichhybridizes under stringent conditions to at least 30 contiguousnucleotides of FIGS. 1, 7, 9, 13 or 14, or having codons substitutedtherefor which encode the same amino acid as a particular codon.Additionally, phosphorylation (e.g., PKA and PKC) domains, as would berecognized by the those skilled in the art are also considered whenproviding a PNS SCP or encoding nucleic acid according to the invention.

[0044] Percent homology or identity can be determined, for example, bycomparing sequence information using the GAP computer program version6.0, available from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman(Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) which are similar, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unitary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745(1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEINSEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap; and (3) no penalty for end gaps. Ina preferred embodiment, the peptide of the invention corresponds to a SCbiologically active portion of SEQ ID NO:2, or variant thereof e.g., aspresented in FIG. 11A-D.

[0045] Thus, one of ordinary skill in the art, given the teachings andguidance presented in the present specification, will know how to add,delete or substitute other amino acid residues in other positions of aSC to obtain a PNS SCP, including substituted, deletional or additionalvariants, e.g. with a substitution as presented in Tables 1 or 2 below.

[0046] A PNS SCP of the invention also includes a variant wherein atleast one amino acid residue in the peptide has been conservativelyreplaced, added or deleted by at least one different amino acid. For adetailed description of protein chemistry and structure, See, e.g.,Schulz, et al., Principles of Protein Structure, Springer-Verlag, N.Y.,1978, and Creighton, T. E., Proteins, Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. F r a presentation of nucleotide sequencesubstitutions, such as codon preferences, see Ausubel et al., eds,Current Protocols in Molecular Biology, Greene Publishing Assoc., NewYork, N.Y. (1987, 1992, 1993, 1994, 1995) at §§ A. 1.1-A.1.24, andSambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), at AppendicesC and D.

[0047] Conservative substitutions of a PNS SCP of the invention includesa variant wherein at least one amino acid residue in the peptide hasbeen conservatively replaced, added or deleted by at least one differentamino acid. Such substitutions preferably are made in accordance withthe following list as presented in Table 1, which substitutions can bedetermined by routine experimentation to provide modified structural andfunctional properties of a synthesized peptide molecule, whilemaintaining SC biological activity, as determined by known SC activityassays. In the context of the invention, the term PNS SCP or“substantially corresponding to” includes such substitutions. TABLE 1Original Exemplary Residue Substitution Ala Gly; Ser Arg Lys Asn Gln;His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu;Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; TyrSer Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0048] Alternatively, another group of substitutions of PNS SCPs of theinvention are those in which at least one amino acid residue in theprotein molecule has been removed and a different residue added in itsplace according to the following Table 2. The types of substitutionswhich can be made in the protein or peptide molecule of the inventioncan be based on analysis of the frequencies of amino abid changesbetween a homologous protein of different species, such as thosepresented in Table 1-2 of Schultz et al., infra. Based on such ananalysis, alternative conservative substitutions are defined herein asexchanges within one of the following five groups: TABLE 2 1. Smallaliphatic. nonpolar or slightly polar residues: Ala. Ser, Thr (Pro,Gly); 2. Polar, negatively charged residues and their amides: Asp, Asn,Glu, Gln; 3. Polar, positively charged residues: His, Arg, Lys; 4. Largealiphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Largearomatic residues: Phe. Tyr. Trp.

[0049] Most deletions and additions, and substitutions according to theinvention are those which do not produce radical changes in thecharacteristics of the protein or peptide molecule. “Characteristics” isdefined in a non-inclusive manner to define both changes in secondarystructure, e.g. α-helix or β-sheet, as well as changes in physiologicalactivity, e.g. in receptor binding assays.

[0050] Accordingly, based on the above examples of specificsubstitutions, alternative substitutions can be made by routineexperimentation, to provide alternative PNS SCPs of the invention, e.g.,by making one or more conservative substitutions of SC fragments whichprovide SC activity. However, when the exact effect of the substitution,deletion, or addition is to be confirmed, one skilled in the art willappreciate that the effect of at least one substitution, addition ordeletion will be evaluated by at least one sodium channel activityscreening assay, such as, but not limited to, immunoassays or bioassays,to confirm biological activity, such as, but not limited to, sodiumchannel activity.

[0051] Amino acid sequence variants of a PNS SCP of the invention canalso be prepared by mutations in the DNA. Such variants include, forexample, deletions from, or additions or substitutions of, residueswithin the amino acid sequence. Any combination of deletion, addition,and substitution can also be made to arrive at the final construct,provided that the final construct possesses some SC activity. Preferablyimproved SC activity is found over that of the non-variant peptide.Obviously, mutations that will be made in the DNA encoding the variantmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNA structure(see, e.g., EP Patent Application Publication No. 75,444; Ausubel,infra; Sambrook, infra). At the genetic level, these variants ordinarilyare prepared by site-directed mutagenesis of nucleotides in the DNAencoding a PNS SCP, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring SC (see, e.g., Ausubel, infra; Sambrook, infra).

[0052] Once a PNS sodium channel structure or characteristics have beendetermined, PNS SCPs can be recombinantly or synthetically produced, oroptionally purified, to provide commercially useful amounts of PNS SCPsfor use in diagnostic or research applications, according to knownmethod steps (see, e.g., Ausubel, infra, and Sambrook, infra, whichreferences are herein entirely incorporated by reference).

[0053] A variety of methodologies known in the art can be utilized toobtain an isolated PNS SCP of the invention. In one embodiment, thepeptide is purified from tissues or cells which naturally produce thepeptide. Alternatively the above-described isolated nucleic acidfragments could be used to expressed the PNS SCP protein in anyorganism. The samples of the invention include cells, protein extractsor membrane extracts of cells, or biological fluids. The sample willvary based on the assay format, the detection method and the nature ofthe tissues, cells or extracts used as the sample.

[0054] The cells and/or tissue can include, e.g., normal or pathologicanimal cells or tissues, such as the peripheral nervous system, andextracts or cell cultures thereof, provided in vivo, in situ or invitro, as cultured, passaged, non-passaged, transformed, recombinant, orisolated cells and/or tissues.

[0055] Any higher eukaryotic organism can be used as a source of atleast one PNS SCI or PNS SCP of the invention, as long as the sourceorganism naturally contains such a peptide. As used herein, “sourceorganism” refers to the original organism from which the amino acidsequence of die peptide is derived, regardless of the organism thepeptide is expressed in and/or ultimately isolated from. Preferredorganisms as sources of at least one PNS SCI or encoding nucleic acidcan be any vertebrate animal, such as mammals, birds, bonyfish,,electric eels, frogs and toads. Among mammals, the preferredrecipients are mammals of the Orders Primata (including humans, apes andmonkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs),Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora(including cats, and dogs). The most preferred source organisms arehumans.

[0056] One skilled in the art can readily follow known methods forisolating proteins in order to obtain the peptide free of naturalcontaminants. These include, but are not limited to:immunochromotography, size-exclusion chromatography, HPLC, ion-exchangechromatography, and immunoaffinity chromatography. See, e.g., Ausubel,infra; Sambrook, infra; Colligan, infra.

[0057] Isolated Nucleic Acid Molecules Coding for PNS SCP Peptides Inone embodiment, the present invention relates to an isolated nucleicacid molecule coding for a peptide having an amino acid sequencecorresponding to novel PNS SCPs. In one preferred embodiment, theisolated nucleic acid molecule comprises a PNS SCP nucleotide sequencewith greater than 70% overall identity or homology to. at least a 60nucleotide sequence present in SEQ ID NO:1 (preferably greater than 80%;more preferably greater than 90%, such as 70-99% any range or valuetherein). In another preferred embodiment, the isolated nucleic acidmolecule comprises a PNS SCP nucleotide sequence corresponding to FIGS.1, 7 or 9, or encoding at least one domain of FIGS. 1, 7, 8, 10 and 11.

[0058] Also included within the scope of this invention are thefunctional equivalents of the herein-described isolated nucleic acidmolecules and derivatives thereof. For example, as presented above forPNS SCP amino acid sequences, the nucleic acid sequences depicted in SEQID NO:1 can be altered by substitutions, additions or deletions thatprovide for functionally equivalent molecules. Due to the degeneracy ofnucleotide coding sequences, other DNA sequences which encodesubstantially the same amino acid sequence of a PNS SCP can be used inthe practice of the invention. These include but are not limited toamino acid sequences encoding all or portions of PNS SCP amino acidsequence of FIGS. 1, 8, 10 and 11, which are altered by the substitutionof different codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change.

[0059] Such functional alterations of a given nucleic acid sequenceafford an opportunity to promote secretion and/or processing ofheterologous proteins encoded by foreign nucleic acid sequences fusedthereto. All variations of the nucleotide sequence of the PNS SCP geneand fragments thereof permitted by the genetic code are, therefore,included in this invention. See, e.g., Ausubel, infra, Sambrook, infra.

[0060] In addition, the nucleic acid sequence can comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of a nucleic acidsequence corresponding to FIGS. 1, 7 or 9, or encoding at least aportion of FIGS. 1, 8, 10 or 11, or a variant thereof. Any nucleotide orpolynucleotide can be used in this regard, provided that its addition,deletion or substitution does remove the sodium channel activity whichis encoded by the nucleotide sequence. Moreover, the nucleic acidmolecule of the invention can, as necessary, have restrictionendonuclease recognition sites which do not remove the activity of theencoded PNS SCP.

[0061] Further, it is possible to delete codons or to substitute one ormore codons by codons other than degenerate codons to produce astructurally modified peptide, but one which has substantially the sameutility or activity of the peptide produced by the unmodified nucleicacid molecule. As recognized in the art, the two peptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode. See, e.g., Ausubel, infra; Sambrook, infra.

[0062] Isolation of Nucleic Acid In another aspect of the presentinvention, isolated nucleic acid molecules coding for peptides havingamino acid sequences corresponding to PNS SCP are provided. Inparticular, the nucleic acid molecule can be isolated from a biologicalsample containing mammalian nucleic acid, as corresponding to a probespecific for a PNS SC obtained from a higher eukaryotic organism.

[0063] The nucleic acid molecule can be isolated from a biologicalsample containing nucleic acid using known techniques, such as but notlimited to, primer amplification or cDNA cloning.

[0064] The nucleic acid molecule can be isolated from a biologicalsample containing genomic DNA or from a genomic library. Suitablebiological samples include, but are not limited to, normal or pathologicanimal cells or tissues, such as cerebrospinal fluid (CNS), peripheralnervous system (neurons, ganglion) and portions, cells of heart, smooth,skeletal or cardiac muscle, autonomic nervous system, and extracts orcell cultures thereof, provided in vivo, in situ or in vitro, ascultured, passaged, non-passaged, transformed, recombinant, or isolatedcells and/or tissues. The method of obtaining the biological sample willvary depending upon the nature of the sample.

[0065] One skilled in the art will realize that a mammalian genome canbe subject to slight allelic variations between individuals. Therefore,the isolated nucleic acid molecule is also intended to include allelicvariations, so long as the sequence encodes a PNS SCP. When a PNS SCPallele does not encode the identical amino acid sequence to that foundin FIGS. 1, 8, 10 or 11, or at least domain thereof, it can be, isolatedand identified as PNS SCP using the same techniques used herein, andespecially nucleic acid amplification techniques to amplify theappropriate gene with primers based on the sequences disclosed herein.Such variations are presented, e.g., in FIG. 11 and in Tables 1 and 2.

[0066] The cloning of large cDNAs is the same (e.g., PN1 as a PNS SCP ofthe invention includes overlapping clones of about 13 kDa) but takesmore routine experimentation, than smaller cDNAs. One useful methodrelies on cDNA bacteriophage library screening. (see, e.g., Sambrook,infra, or Ausubel, infra). Probes for the screening are labeled, e.g.,with random hexamers and Klenow enzyme (Pharmacia kit). If 5′ cDNAs arenot obtained with these approaches, a subcDNA library can be prepared inwhich a specific PN1 primers are used to prime the reverse transcriptreaction in place of oligo dT or random primers. The cDNA sublibrary isthen cloned into standard vectors such as lambda zap and screened usingconventional techniques. This strategy was used previously (Noda et al.Nature 320:188-192 (1986); Noda et al., Nature 322:826-828 (1986)) toclone the brain type I and II sodium channel cDNAs. The construction ofa full-length cDNA is performed by subcloning overlapping fragments intoan expression vector (either prokaryotic or eukaryotic). This task ismore difficult with large cDNAs because of the paucity of uniquerestriction sites, but routine restriction, cloning or PCR is used tojoin the fragments.

[0067] Synthesis of Nucleic Acid Isolated nucleic acid molecules of thepresent invention are also meant to include those chemically synthesizedFor example, a nucleic acid molecule with the nucleotide sequence whichcodes for the expression product of a PNS SCP gene can be designed and,if necessary, divided into appropriate smaller fragments.

[0068] Then an oligomer which corresponds to the nucleic acid molecule,or to each of the divided fragments, can be synthesized (e.g., of10-6015 nucleotides or any range or value therein, such as 10-100nucleotides). Such synthetic oligonucleotides can be prepared, forexample, by known techniques (See, e.g., Ausubel, infra, or Sambrook,infra) or by using an automated DNA synthesizer.

[0069] A labeled oligonucleotide probe be derived synthetically or bycloning. If necessary, the 5′-ends of the oligomers can bephosphorylated using T4 polynucleotide kinase. Kinasing of singlestrands prior to annealing or for labeling can be achieved using anexcess of the enzyme. If kinasing is for the labeling of probe, the ATPcan contain high specific activity radioisotopes. Then, the DNA oligomercan be subjected to annealing and ligation with T4 ligase or the like.

[0070] A Nucleic Acid Probe for the Specific Detection of PNS SCP Inanother embodiment, the present invention relates to a nucleic acidprobe of 15-6000 nucleotides for the specific detection of the presenceof PNS SCP in a sample comprising the above-described nucleic acidmolecules or at least a fragment thereof which binds under stringentconditions to a nucleic acid encoding at least one PNS SCP.

[0071] The nucleic acid probe can be used to screen an appropriatechromosomal or cDNA library by known hybridization method steps toobtain a PNS SCP encoding nucleic acid molecule of the invention. Achromosomal DNA or cDNA library can be prepared from appropriate cellsaccording to recognized methods in the art (See, e.g., Ausubel, infra;Sambrook, infra).

[0072] In the alternative, organic chemical synthesis is carried out inorder to obtain nucleic acid probes having nucleotide sequences whichcorrespond to suitable portions of the amino acid sequence of the PNSSCP. Thus, the synthesized nucleic acid probes can be used as primers innucleic acid amplification method steps

[0073] The invention can thus provide methods for amplification of DNAand/or RNA using heat stable, cross-linked nucleotide primers, whichcross linked primers of the invention to provide nucleic acid encodingPNS SCPs according to the invention.

[0074] Methods of amplification of RNA or DNA are well known in the artand can be used according to the invention without undueexperimentation, based on the teaching and guidance presented herein.According to the invention, the use of nucleic acids encoding portionsof PNS SCPs according to the invention, as amplification primers, allowsfor advantages over known amplification primers, due to the increase insensitivity, selectivity and/or rate of amplification.

[0075] Known methods of DNA or RNA amplification include, but are notlimited to polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson et al.; U.S. Pat. No. 5,091,310 to Inn U.S. Pat. No. 5,066,584to Gyllensten et al.; U.S. Pat. No. 4,889,818 to Gelfand et al; U.S.Pat. No. 4,994,370 to Silver et al.; U.S. Pat. No. 4,766,067 to Biswas;U.S. Pat. No. 4,656,134 to Ringold; U.S. Pat. No. 5,340,728 to Grosz etal.; U.S. Pat. No. 5,322,770 to Gelfand et al.; U.S. Pat. No. 5,338,671to Scalice et al.; PCT WO 92/06200 to Cetus Corp.; PCT WO 94/14978 toStrack et al., which patent disclosures are entirely incorporated hereinby reference) and RNA mediated amplification which uses antisense RNA tothe target sequence as a template for double stranded DNA synthesis(U.S. Pat. No. 5,130,238 to Malek et al., with the tradeneame NASBA),the entire contents of which patents and references are herein entirelyincorporated by reference. Reviews of the PCR are provided by Mullis(Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki et al.(Bio/Technology 3:1008-1012 (1985)); and Mullis et al. (Meth Enzyrol.155:335-350 (1987)). One skilled in the art can readily design suchprobes based on the sequence disclosed herein using methods such ascomputer alignment and sequence analysis known in the art. See, e.g.,Ausubel, infra, Sambrook, infra.

[0076] The hybridization probes of the invention can be labeled bystandard labeling techniques such as with a radiolabel, enzyme label,fluorescent label, biotin-avidin label, chemiluminescence, and any otherknown and suitable labels. After hybridization, the probes can bevisualized using known methods. The nucleic acid probes of the inventioninclude RNA, as well as DNA probes, such probes being generated usingtechniques known in the art (See, e.g., Ausubel, infra; Sambrook,infra). In one embodiment of the above described method, a nucleic acidprobe is immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and SEPHAROSE, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art (See, e.g.,Ausubel, infra; Sambrook, infra).

[0077] The test samples suitable for nucleic acid probing methods of theinvention include, for example, cells or nucleic acid extracts of cells,or biological fluids. The sample used in the above-described methodswill vary based on the assay format, the detection method and the natureof the tissues, cells or extracts to be assayed. Methods for preparingnucleic acid extracts of cells are well known in the art and can bereadily adapted in order to obtain a sample which is compatible with themethod utilized.

[0078] Methods for Detecting The Presence of PNS SCP Encoding NucleicAcid in a Biological Sample. In another embodiment, the presentinvention relates to methods for detecting the presence of PNS SCPencoding nucleic acid in a sample. Such methods can comprise (a)contacting the sample with the above-described nucleic acid probe, underconditions such that hybridization occurs, and (b) detecting thepresence of a labeled probe bound to the nucleic acid probe. One skilledin the art can select a suitable, labeled nucleic acid probe accordingto techniques known in the art as described above. Samples to be testedinclude, but are not limited to, RNA samples of mammalian tissue.

[0079] PNS SCP has been found to be expressed in peripheral nerve anddorsal root ganglion cells. Accordingly, PNS SCP probes can be useddetect the presence of RNA from PN cells in such a biological sample.Further, altered expression levels of PNS SCP RNA in an individual, ascompared to normal levels, can indicate the presence of disease. The PNSSCP probes cart further be used to assay cellular activity in generaland specifically in peripheral nervous system tissue.

[0080] A Kit for Detecting the Presence of PNS SCP in a Sample. Inanother embodiment, the present invention relates to a kit for detectingthe presence of PNS SCP in a sample comprising at least one containerhaving disposed therein the above-described nucleic acid probe. In apreferred embodiment, the kit further comprises other containerscomprising one or more of the following: wash reagents and reagentscapable of detecting the presence of bound nucleic acid probe. Examplesof detection reagents include, but are not limited to radiolabeledprobes, enzymatic labeled probes (horse radish peroxidase, allalinephosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin) (See, e.g., Ausubel, infra, Sambrook, infra).

[0081] A compartmentalized kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, TRIS-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like.

[0082] One skilled in the art will readily recognize that the nucleicacid probes described in the invention can readily be incorporated intoone of the established kit formats which are well known in the art.

[0083] DNA Constructs Comprising a PNS SCP Nucleic Acid Molecule andHosts Containing These Constructs. A nucleic acid sequence encoding anPNS SCP of the invention can be recombined with vector DNA in accordancewith conventional techniques, including blunt-ended or staggered-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, e.g., by Ausubel et al., infra, and are well known in theart.

[0084] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as PNS SCPs or Abfragments in recoverable amounts. The precise nature of the regulatoryregions needed for gene expression can vary from organism to organism,as is well known in the analogous art. See, e.g., Sambrook, infra andAusubel infra.

[0085] The invention accordingly encompasses the expression of an PNSSCP, in either prokaryotic or eukaryotic cells, although eukarypticexpression is preferred.

[0086] Preferred hosts are bacterial or eukaryotic hosts includingbacteria, yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. It is preferred that the mammalian cell or tissue is of human,primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog orcat origin, but any other mammalian cell can be used.

[0087] Eukaryotic hosts can include yeast, insects, fungi, and mammaliancells either in vivo, or in tissue culture. Preferred eukaryotic hostscan also include, but are not limited to insect cells, mammalian cellseither in vivo, or in tissue culture. Preferred mammalian cells includeXenopus oocytes, HeLa cells, cells of fibroblast origin such as VERO orCHO-K1, or cells of lymphoid origin and their derivatives.

[0088] Mammalian cells provide post-translational modifications toprotein molecules including correct folding or glycosylation at correctsites. Mammalian cells which can be useful as hosts include cells offibroblast origin such as, but not limited to, NIH 3T3, VERO or CHO, orcells of lymphoid origin, such as, but not limited to, the hybridomaSP2/O-Ag14 or the murine myeloma P3-X63Ag8, hamster cell lines (e.g.,CHO-K1 and progenitors, e.g., CHO-DUXB11) and their derivatives. Onepreferred type of mammalian cells are cells which are intended toreplace the function of the genetically deficient cells in vivo.Neuronally derived cells are preferred for gene therapy of disorders ofthe nervous system. For a mammalian cell host, many possible vectorsystems are available for the expression of at least one PNS SCP. A widevariety of transcriptional and translational regulatory sequences can beemployed, depending upon the nature of the host. The transcriptional andtranslational regulatory signals can be derived from viral sources, suchas, but not limited to, adenovirus, bovine papilloma virus, Simianvirus, or the like, where the regulatory signals are associated with aparticular gene which has a high level of expression. Alternatively,promoters from mammalian expression products, such as, but not limitedto, actin, collagen, myosin, protein production. See, Ausubel, infra,;Sanbrook, infra.

[0089] When live insects are to be used, silk moth caterpillars andbaculoviral vectors are presently preferred hosts for large scale PNSSCP production according to the invention. Production of PNS SCPs ininsects can be achieved, for example, by infecting the insect host witha baculovirus engineered to express at least one PNS SCP by methodsknown to those skilled in the related arts. See Ausubel et al, eds.Current Protocols in Molecular Biology, Wiley Interscience, §§16.8-16.11(1987, 1992, 1993, 1994).

[0090] In a preferred embodiment, the introduced nucleotide sequencewill be incorporated into a plasmid or viral vector capable ofautonomous replication in the recipient host. Any of a wide variety ofvectors can be employed for this purpose. See, e.g., Ausubel et al.,infra, §§ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and16.8-16.11. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector can be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

[0091] Different host cells have characteristic and specific mechanismsfor the translational and post-translational processing and modification(e.g., glycosylation, cleavage) of proteins. Appropriate cell lines orhost systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an unglycosylated core proteinproduct. Expression in yeast will produce a glycosylated product.Expression in mammalian cells can be used to ensure “native”glycosylation of the heterologous PNS SCP protein. Furthermore,different vector/host expression systems can effect processing reactionssuch as proteolytic cleavages to different extents.

[0092] As discussed above, expression of PNS SCP in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. See, e.g., Ausubel, infra, Sambrook, infra.

[0093] Once the vector or nucleic acid molecule containing theconstruct(s) has been prepared for expression, the DNA construct(s) canbe introduced into an appropriate host cell by any of a variety ofsuitable means, i.e., transformation, transfection, conjugation,protoplast fusion, electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofat least one PNS SCP. This can take place in the transformed cells assuch, or following the induction of these cells to differentiate (forexample, by administration of bromodeoxyuracil to neuroblastoma cells orthe like).

[0094] Isolation of PNS SCP. The PNS SCP proteins or fragments of thisinvention can be obtained by expression from recombinant DNA asdescribed above. Alternatively, a PNS SCP can be purified frombiological material. If so desired, the expressed at least one PNS SCPcan be isolated and purified in accordance with conventional methodsteps, such as extraction, precipitation, chromatography, affinitychromatography, electrophoresis, or the like. For example, cellsexpressing at least one PNS SCP in suitable levels can be collected bycentrifugation, or with suitable buffers, lysed, and the proteinisolated by column chromatography, for example, on DEAE-cellulose,phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or byelectrophoresis or immunoprecipitation. Alternatively, PNS SCPs can beisolated by the use of specific antibodies, such as, but not limited to,an PNS SCP or SC antibody. Such antibodies can be obtained by knownmethod steps (see, e.g. Colligan, infra; Ausubel, infra.

[0095] For purposes of the invention, one method of purification whichis illustrative, without being limiting, consists of the followingsteps. A fist step in the purification of a PNS SCP includes extractionof the PNSSCP fraction from a biological sample, such as peripheralnerve tissue or dorsal root ganglia (DRG), in buffers, with or withoutsolubilizing agents such as urea, formic acid, detergent, orthiocyanate. A second step includes subjecting the solubilized materialto ion-exchange chromatography on Mono-Q-or Mono-S columns (PharmaciaLKB Biotechnology, Inc; Piscataway, N.J.). Similarly, the solubilizedmaterial can be separated by any other process wherein molecules can beseparated according to charge density, charge distribution and molecularsize, for example. Elution of the PNS SCP from the ion-exchange resinare monitored by an immunoassay, such as M-IRMA, on each fraction.Immunoreactive peaks would are then dialyzed, lyophilized, and subjectedto molecular sieve, or gel chromatography. In a third step, molecularsieve or gel chromatography is a type of partition chromatography inwhich separation is based on molecular size. Dextran, polyacrylamide,and agarose gels are commonly used for this type of separation. Oneuseful gel for the invention is SEPHAROSE 12 (Pharmacia LKBBiotechnology, Inc.). However, other methods, known to those of skill inthe art can be used to effectively separate molecules based on size. Afourth step in a purification protocol for a PNS SCP can includeanalyzing the immunoreactive peaks by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a further gelchromatographic purification step, and staining, such as, for example,silver staining. A fifth step in a purification method can includesubjecting the PNS SCP obtained after SDS-PAGE to affinitychromatography, or any other procedure based upon affinity between asubstance to be isolated and a molecule to which it can specificallybind. For further purification of a PNS SCP, affinity chromatography onSEPHAROSE conjugated to anti-PNS SCP mAbs (specific mABs generatedagainst substantially pure PNS SCP) can be used. Alternative methods,such as reverse-phase HPLC, or any other method characterized by rapidseparation with good peak resolution are useful.

[0096] It will be appreciated that other purification steps can besubstituted for the preferred method described above. Those of skill inthe art will be able to devise alternate purification schemes withoutundue experimentation.

[0097] An Antibody Having Binding Affinity to a PNS SCP Peptide and aHybridoma Containing the Antibody. In another embodiment, the inventionrelates to an antibody having binding affinity specifically to a PNS SCPpeptide as described above or fragment thereof. Those which bindselectively to PNS SCP would be chosen for use in methods which couldinclude, but should not be limited to, the analysis of altered PNS SCPexpression in tissue containing PNS SCP.

[0098] The PNS SCP proteins of the invention can be used in a variety ofprocedures and methods, such as for the generation of antibodies, foruse in identifying pharmaceutical compositions, and for studyingDNA/protein interaction.

[0099] The PNS SCP peptide of the invention can be used to produceantibodies or hybridomas. One skilled in the art will recognize that ifan antibody is desired, such a peptide would be generated as describedherein and used as an immunogen.

[0100] The antibodies of the invention include monoclonal and polyclonalantibodies, as well as fragments of these antibodies. The inventionfurther includes single chain antibodies. Antibody fragments whichcontain the idiotype of the molecule can be generated by knowntechniques.

[0101] The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic(anti-Id) antibodies to antibodies that can be labeled in soluble orbound form, as well as fragments thereof provided by any knowntechnique, such as, but not limited to enzymatic cleavage, peptidesynthesis or recombinant techniques. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofanimals immunized with an antigen. A monoclonal antibody contains asubstantially homogeneous population of antibodies specific to antigens,which population contains substantially similar epitope binding sites.MAbs can be obtained by methods known to those skilled in the art. See,e.g., Kohler and Milstein, Nature 256:495-497 (1975); U.S. Pat. No.4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); andHarlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring HarborLaboratory (1988); Colligan et al., eds., Current Protocols inImmunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), the contents of which references are incorporated entirelyherein by reference. Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridomaproducing a mAb of the invention can be cultivated in vitro, in situ orin vivo. Production of high titers of mAbs in vivo or in situ makes thisthe presently preferred method of production.

[0102] Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine mAb and a human immunoglobulin constantregion, which are primarily used to reduce immunogenicity in applicationand to increase yields in production, for example, where murine mabshave higher yields from hybridomas but higher immunogenicity in humans,such that human/murine chimeric mAbs are used. Chimeric antibodies andmethods for their production are known in the art (Cabilly et al, Proc.Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-855 (1984); Boulianne et al., Nature 312:643-646(1984); Cabilly et al, European Patent Application 125023; Neuberger etal., Nature 314:268-270 (1985); Taniguchi et al., European PatentApplication 171 496; Morrison et al., European Patent Application 173494; Neuberger et al, PCT Application WO 86/01533; Kudo et al., EuropeanPatent Application 184 187; Morrison et al., European Patent Application173 494, Sahagan et al., J. Immunol. 137:1066-1074.(1986); Robinson etal., International Patent Publication No. PCT/US86/02269; Liu et al.,Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al, Proc. Natl.Acad. Sci. USA 84:214-218 (1987); Better et al, Science 240:1041-1043(1988); and Harlow, infra. These references are entirely incorporatedherein by reference.

[0103] An anti-idiotypic (anti-Id) antibody is an antibody whichrecognizes unique determinants generally associated with theantigen-binding site of an antibody. An Id antibody can be prepared byimmunizing an animal of the same species and genetic type (e.g., mousestr) as the source of the mAb with the mAb to which an anti-Id is beingprepared. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing antibody by producing anantibody to these idiotypic determinants (the anti-Id antibody). See,for example, U.S. Pat. No. 4,699,880, which is herein entirelyincorporated by reference.

[0104] The anti-Id antibody can also be used as an “immunogen” to inducean immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id can be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

[0105] Accordingly, mAbs generated against a PNS SCP of the inventioncan be used to induce anti-Id antibodies in suitable animals, such asBALB/c mice. Spleen cells from such immunized mice are used to produceanti-Id hybridomas secreting anti-Id mabs. Further, the anti-Id mAbs canbe coupled to a carrier such as keyhole limpet hemocyanin (KLH) and usedto immunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for a PNS SCP specific epitope. The anti-Id mabs thus havetheir own idiotypic epitopes, or “idiotopes” structurally similar to theepitope being evaluated.

[0106] The term “antibody” is also meant to-include both intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab′)₂, which are capable of binding antigen. Fab and F(ab′)₂ fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and can have less non-specific tissue binding than anintact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It willbe appreciated that Fab and F(ab′)₂ and other fragments of theantibodies useful in the invention can be used for the detection and/orquantitation of a PNS SCP according to the methods disclosed herein forintact antibody molecules. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). An antibody is saidto be “capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.The term “epitope” is meant to refer to that portion of any moleculecapable of being bound by an antibody which can also be recognized bythat antibody. Epitopes or “antigenic determinants” usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structuralcharacteristics as well as specific charge characteristics.

[0107] An “antigen” is a molecule or a portion of a molecule capable ofbeing bound by an antibody which is additiolially capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen can have one, or more than one epitope. The specificreaction referred to above is meant to indicate that the antigen willreact, in a highly selective manner, with its corresponding antibody andnot with the multitude of other antibodies which can be evoked by otherantigens.

[0108] Immunoassays. Antibodies of the invention, directed against a PNSSCP, can be used to detect or diagnose a PNS SC or a PNS SC-relatedpathologies. Screening methods are provided by the invention caninclude, e.g., immunoassays employing radioimmunoassay (RIA) orenzyme-linked immunosorbant assay (ELISA) methodologies, based on theproduction of specific antibodies (monoclonal or polyclonal) to a PNSSCP. For these assays, biological samples are obtained by, nerve biopsy,or other peripheral nervous system tissue sampling. For example, in oneform of RIA, the substance under test is mixed with diluted antiserum inthe presence of radiolabeled antigen. In this method, the concentrationof the test substance will be inversely proportional to the amount oflabeled antigen bound to the, specific antibody and directly related tothe amount of free labeled antigen. Other suitable screening methodswill be readily apparent to those of skill in the art.

[0109] Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides”, In: SyntheticPeptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992), andKaspczak et al., Biochemistry 28:9230-8 (1989).

[0110] One embodiment for carrying out the diagnostic assay of theinvention on a biological sample containing a PNS SCP, comprises:

[0111] (a) contacting a detectably labeled PNS SCP-specific antibodywith a solid support to effect immobilization of said PNS SCP-specificantibody or a fragment thereof;

[0112] (b) contacting a sample suspected of containing a PNS SCP withsaid solid support;

[0113] (c) incubating said detectably labeled PNS SCP-specific antibodywith said support for a time sufficient to allow the immobilized PNSSCP-specific antibody to bind to the PNS SCP;

[0114] (d) separating the solid phase support from the incubationmixture obtained in step (c); and

[0115] (e) detecting the bound label and thereby detecting andquantifying PNS SCP.

[0116] The specific concentrations of detectably labeled antibody andPNS SCP, the temperature and time of incubation, as well as other assayconditions can be varied, depending on various factors including theconcentration of a PNS SCP in the sample, the nature of the sample, andthe like. The binding activity of a given lot of anti-PNS SCP antibodycan be determined according to well known methods. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation. Other suchsteps as washing, stirring, shaking, filtering and the like can be addedto the assays as is customary or necessary for the particular situation.

[0117] Detection can be accomplished using any of a variety of assays.For example, by radioactively labeling the PNS SCP-specific antibodiesor antibody fragments, it is possible to detect PNS SCP through the useof radioimmune assays. A good description of a radioimmune assay can befound in Colligan, infra, and Ausubel, infra, entirely incorporated byreference herein. Preferably, the detection of cells which express a PNSSCP can be accomplished by in vivo imaging techniques, in which thelabeled antibodies (or fragments thereof) are provided to a subject, andthe presence of the PNS SCP is detected without the prior removal of anytissue sample. Such in vivo detection procedures have the advantage ofbeing less invasive than other detection methods, and are, moreover,capable of detecting the presence of PNS SCP in tissue which cannot beeasily removed from the patient, such as brain tissue.

[0118] There are many different in vivo labels and methods of labelingknown to those of ordinary skill in the art Examples of the types oflabels which can be used in the invention include radioactive isotopesand paramagnetic isotopes. Those of ordinary skill in the art will knowof other suitable labels for binding to the antibodies used in theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to theantibodies can be done using standard techniques common to those ofordinary skill in the art.

[0119] For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay which is detectable for agiven type of instrument In general, any conventional method forvisualling diagnostic imaging can be utilized in accordance with thisinvention. For example, positron emission tomography (PET), gamma, beta,and magnetic resonance imaging (MRI) detectors can be used to visualizediagnostic imagining.

[0120] The antibodies useful in the invention can also be labeled withparamagnetic isotopes for purposes of in vivo diagnosis. Elements whichare particularly useful, as in Magnetic Resonance Imaging (MRI), include¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, and ⁵⁶Fe.

[0121] The antibodies (or fragments thereof) useful in the invention arealso particularly suited for use in in vitro immunoassays to detect thepresence of a PNS SCP in body tissue, fluids (such as CSF), or cellularextracts. In such immunoassays, the antibodies (or antibody fragments)can be utilized in liquid phase or, preferably, bound to a solid-phasecarrier, as described above.

[0122] In situ detection can be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledantibodies of the invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof a PNS SCP, but also the distribution of a PNS SCP on the examinedtissue. Using the invention, those of ordinary skill will readilyperceive that any of a wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0123] As used herein, an effective amount of a diagnostic reagent (suchas an antibody or antibody fragment) is one capable of achieving thedesired diagnostic discrimination and will vary depending on suchfactors as age, condition, sex, the extent of disease of the subject,counter-indications, if any, and other variables to be adjusted by thephysician. The amount of such materials which are typically used in adiagnostic test are generally between 0.1 to 5 mg, and preferablybetween 0.1 to 0.5 mg.

[0124] The assay of the invention is also ideally suited for thepreparation of a kit. Such a kit can comprise a carrier means beingcompartmentalized to receive in close confinement therewith one or morecontainer means such as vials, tubes and the like, each of saidcontainer means comprising the separate elements of the immunoassay.

[0125] For example, there can be a container means containing a first-antibody immobilized on a solid phase support, and a further containermeans containing a second detectably labeled antibody in solution.Further container means can contain standard solutions comprising serialdilutions of the PNS SCP to be detected. The standard solutions of a PNSSCP can be used to prepare a standard curve with the concentration ofPNS SCP plotted on the abscissa and the detection signal on theordinate. The results obtained from a sample containing a PNS SCP can beinterpolated from such a plot to give the concentration of the PNS SCP.

[0126] Diagnostic Screening and Treatment It is to be understood thatalthough the following discussion is specifically directed to humanpatients, the teachings are also applicable to any animal that expressesat least one PNS SC. The diagnostic and screening methods of theinvention are especially useful for a patient suspected of being at riskfor developing a disease associated with an altered expression level ofPNS SCP based on family history, or a patient in which it is desired todiagnose a PNS SCP-related disease.

[0127] According to the invention, presymptomatic screening of anindividual in need of such screening is now possible using DNA encodingthe PNS SCP protein of the invention. The screening method of theinvention allows a presymptomatic diagnosis, including prenataldiagnosis, of the presence of a missing or aberrant PNS SC gene inindividuals, and thus an opinion concerning the likelihood that suchindividual would develop or has developed a PNS SC-associated disease.This is especially valuable for the identification of carriers ofaltered or missing PNS SC genes, for example, from individuals with afamily history of a PNS SC-related pathology. Early diagnosis is alsodesired to maximize appropriate timely intervention.

[0128] In one preferred embodiment of the method of screening, a tissuesample would be taken from such individual, and screened for (1) thepresence of the “normal” PNS SCP gene; (2) the presence of PNS SCP mRNAand/or (3) the presence of PNS SCP protein. The normal human gene can becharacterized based upon, for example, detection of restrictiondigestion patterns in “normal” versus the patient's DNA, including RFLPanalysis, using DNA probes prepared against the PNS SCP sequence (or afunctional fragment thereof) taught in the invention. Similarly, PNS SCPmRNA can be characterized and compared to normal PNS SCP mRNA (a) levelsand/or (b) size as found in a human population not at risk of developingPNS SCP-associated disease using similar probes. Lastly, PNS SCP proteincan be (a) detected and/or (b) quantitated using a biological assay forPNS SCP activity or using an immunological assay and PNS SCP antibodies.When assaying PNS SCP protein, the immunological assay is preferred forits speed. An (1) aberrant PNS SCP DNA size pattern, and/or (2) aberrantPNS SCP mRNA sizes or levels and/or (3) aberrant PNS SCP protein levelswould indicate that the patient is at risk for developing a PNSSCP-associated disease.

[0129] The screening and diagnostic methods of the invention do notrequire that the entire PNS SCP DNA coding sequence be used for theprobe. Rather, it is only necessary to use a fragment or length ofnucleic acid that is sufficient to detect the presence of the PNS SCPgene in a DNA preparation from a normal or affected individual, theabsence of such gene, or an altered physical property of such gene (suchas a change in electrophoretic migration pattern).

[0130] Prenatal diagnosis can be performed when desired, using any knownmethod to obtain fetal cells, including amniocentesis, chorionic villoussampling (CVS), and fetoscopy. Prenatal chromosome analysis can be usedto determine if the portion of the chromosome possessing the normal PNSSCP gene is present in a heterozygous state.

[0131] Overview of PNS SCP Purification and Crystallization Methods. Ingeneral, a PNS SCP as a membrane protein, is purified in soluble formusing detergents (e.g., octyglucosides) or other suitable amphiphillicmolecules. The resulting PNS SCP is in sufficient purity andconcentration for crystallization. The purified PNS SCP is then isolatedand assayed for biological activity and for lack of aggregation (whichinterferes with crystallization). The purified and cleaved PNS SCPpreferably runs as a single band under reducing or nonreducingpolyacrylamide gel electrophoresis (PAGE) (nonreducing is used toevaluate the presence of cysteine bridges). The purified PNS SCP ispreferably crystallized under varying conditions of at least one of thefollowing: pH, buffer type, buffer concentration, salt type, polymertype, polymer concentration, other precipitating ligands andconcentration of purified and cleaved PNS SCP by known methods. See,e.g., Michel, Trends in Biochem Sci. 8:56-59 (1983); Deisenhofer et al.J. Mol. Biol 180:385-398 (1984); Weiss et al. FEBS Lett. 267:268-272(1990). Blundell, et al. Protein Crystallography Academic Press, London(1976); Oxender et al. eds., Protein Engineering Liss, N.Y. (1986);McPherson; The Preparation and Analysis of Protein Crystals Wiley, N.Y.(1982); or the methods provided in a commercial kit, such as CRYSTALSCREEN (Hampton Research, Riverside, Calif.). The crystallized proteinis also tested for at least one SC activity and differently sized andshaped crystals are further tested for suitability in X-ray diffraction.Generally, larger crystals provide better crystallography than smallercrystals, and thicker crystals provide better crystallography thanthinner crystals. See, e.g., Blundell., infra, Oxender, infra,McPherson, infra; Wyckoff et al, eds., Diffraction Methods forBiological Macromolecules, Vols. 114-115: Methods in Enzymology,Orlando, Fla. Academic Press (1985).

[0132] Protein Crystallization Methods. The hanging drop method ispreferably used to crystallize a purified soluble, PNS SCP protein. See,e.g., Taylor et al., J. Mol. Biol. 226:1287-1290 (1992); Takimoto et al.(1992), infra, CRYSTAL SCREEN, Hampton Research. A mixture of theprotein and precipitant can include the following:  pH (e.g., 4-10); buffer type (e.g., tromethamine (TRIZMA), sodium azide, phosphate,sodium, rcacodylate acetates, imidazole, Tris HCl, sodium hepes); buffer concentration (e.g., 0.1-100 mM);  salt type (e.g., sodiumazide, calcium chloride, sodium citrate, magnesium chloride, ammoniumacetate, ammonium sulfate, potassium phosphate, magnesium acetate, zincacetate; calcium acetate);  polymer type and concentration: (e.g.,polyethylene glycol (PEG) 1-50%, type 6000-10,000);  otherprecipitating ligands (salts: potassium, sodium, tartrate, ammoniumsulfate, sodium acetate, lithium sulfate, sodium formate, sodiumcitrate, magnesium formate, sodium phosphate, potassium phosphage;organics: 2-propanol; non-volatile: 2-methyl-2,4-pentanediol); and concentration of purified PNS SCP (e.g., 0.1-100 mg/ml, with addedamphiphillic molecules (detergents such as octylgluosides)). See, e.g.,CRYSTAL SCREEN, Hampton Research.

[0133] The above mixtures are used and screened by varying at least oneof pH, buffer type; buffer concentration, precipitating salt type orconcentration, PEG type, PEG concentration, and cleaved proteinconcentration. Crystals ranging in size from 0.1-1.5 mm are formed in1-14 days. These crystals diffract X-rays to at least 10 Å resolution,such as 1.5-10.0 Å, or any range of value therein, such as 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 Å or less being preferred for thehighest resolution. In addition to diffraction patterns having thishighest resolution, lower resolution, such as 25-3.5 Å can further beused.

[0134] Protein Crystals. Crystals appear after 1-14 days and continue togrow on subsequent days. Some of the crystals are removed, washed, andassayed for biological activity, which activity is preferred for usingin further characterizations. Other washed crystals are preferably run na stained gel and those that-migrate in the same position as thepurified cleaved PNS SCP are preferably used. From two to one hundredcrystals are observed in one drop and crystal forms can occur, such as,but not limited to, bipyramidal, rhomboid, and cubic. Initial X-rayanalyses are expected to indicate that such crystals diffract atmoderately high to high resolution. When fewer crystals are produced ina drop, they can be much larger size, e.g., 0.2-1.5 mm.

[0135] PNS SCP X-ray Crystallography Methods The crystals so producedfor a PNS SCP are X-ray analyzed using a suitable X-ray source. Asuitable number of diffraction patterns are obtained. Crystals arepreferably stable for at least 10 hrs in the X-ray beam. Frozen crystals(e.g., −220 to −50° C.) are optionally used for longer X-ray exposures(e.g., 4-72 hrs), the crystals being relatively more stable to theX-rays in the frozen state. To collect the maximum number of usefulreflections, multiple frames are optionally collected as the crystal isrotated in the X-ray beam, e.g.; for 12-96 hrs. Larger crystals (>0.2mm) are preferred, to increase the resolution of the X-ray diffraction.Crystals are preferably analyzed using a synchrotron high energy X-raysource. Using frozen crystals, X-ray diffraction data is collected oncrystals that diffract to a resolution of 10-1.5 Å, with lowerresolutions also useful, such as 25-10 Å, sufficient to solve thethree-dimensional structure of a PNS SCP in considerable detail, aspresented herein.

[0136] Computer Related Embodiments. An amino acid sequence of a PNS SCPand/or x-ray diffraction data, useful for computer molecular modeling ofa PNS SCP or a portion thereof, can be “provided” in a variety ofmediums to facilitate use thereof. As used herein, provided refers to amanufacture, which contains a PNS SCP amino acid sequence and/or x-raydiffraction data of the present invention, e.g., the amino sequenceprovided in FIGS. 1, 8, 10 or 11, a representative fragment thereof, oran amino acid sequence having at least 80-100% overall identity to a5-2005 amino acid fragment of an amino acid sequence of FIGS. 11A-D or avariant thereof. Such a method provides the amino acid sequence and/orx-ray diffraction data in a form which allows a skilled artisan toanalyze and molecular model the three dimension structure of a PNS SCPor subdomain thereof.

[0137] In one application of this embodiment, PNS SCP, or at least onesubdomain thereof, amino acid sequence and/or x-ray diffraction data ofthe present invention is recorded on computer readable medium. As usedherein, “computer readable medium” refers to any medium which can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as opticaldiscs or CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Askilled artisan can readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a n amino acid sequenceand/or x-ray diffraction data of the present invention.

[0138] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently know methods for recording information oncomputer readable medium to generate manufactures comprising an aminoacid sequence and/or x-ray diffraction data information of the presentinvention.

[0139] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereonan amino acid sequence and/or x-ray diffraction data of the presentinvention. The choice of the data storage structure will generally bebased on the means chosen to access the stored information. In addition,a variety of data processor programs and formats can be used to storethe sequence and x-ray data information of the present invention oncomputer readable medium. The sequence information can be represented ina word processing text file, formatted in commercially-availablesoftware such as WordPerfect and MicroSoft Word, or represented in theform of an ASCII file, stored in a database application, such as DB2,Sybase, Oracle, or the like. A skilled artisan can readily adapt anynumber of dataprocessor structuring formats (e.g. text file or database)in order to obtain computer readable medium having recorded thereon theinformation of the present invention.

[0140] By providing the PNS SCP sequence and/or x-ray diffraction dataon computer readable medium, a skilled artisan can routinely access thesequence and x-ray diffraction data to model a PNS SCP, a subdomainthereof, or a ligand thereof. Computer algorythms are publicly andcommercially available which allow a skilled artisan to access this dataprovided in a computer readable medium and analyze it for molecularmodeling and/or RDD.

[0141] The present invention further provides systems; particularlycomputer-based systems; which contain the sequence and/or diffractiondata described herein. Such systems are designed to do molecularmodeling and RDD for a PNS SCP or at least one subdomain thereof.

[0142] As used herein, “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thesequence and/or x-ray diffraction data of the present invention. Theminimum hardware means of the computer-based systems of the presentinvention comprises a central processing unit (CPU), input means, outputmeans, and data storage means. A skilled artisan can readily appreciatewhich of the currently available computer-based system are suitable foruse in the present invention.

[0143] As stated above, the computer-based systems of the presentinvention comprise a data storage means having stored therein a PNS SCPor fragment sequence and/or x-ray diffraction data of the presentinvention and the necessary hardware means and software means forsupporting and implementing an analysis means. As used herein, “datastorage means” refers to memory which can store sequence or x-raydiffraction data of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the sequence orx-ray data of the present invention.

[0144] As used herein, “search means” or “analysis means” refers to oneor more programs which are implemented on the computer-based system tocompare a target sequence or target structural motif with the sequenceor x-ray data stored within the data storage means. Search means areused to identify fragments or regions of a PNS SCP which match aparticular target sequence or target motif. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. A skilled artisancan readily recognize that any one of the available algorithms orimplementing software packages for conducting computer analyses that canbe adapted for use in the present computer-based systems.

[0145] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration or electron density map which is formed upon the foldingof the target motif. There are a variety of target motifs known in theart. Protein target motifs include, but are not limited to, enzymicactive sites, structural subdomains, epitopes, functional domains andsignal sequences. A variety of structural formats for the input andoutput means can be used to input and output the information in thecomputer-based systems of the present invention.

[0146] A variety of comparing means can be used to compare a targetsequence or target motif with the data storage means to identifystructural motifs or electron density maps. A skilled artisan canreadily recognize that any one of the publicly available computermodeling programs can be used as the search means for the computer-basedsystems of the present invention.

[0147] One application of this embodiment is provided in FIG. 12. FIG.12 provides a block diagram of a computer system 102 that can be used toimplement the present invention. The computer system 102 includes aprocessor 106 connected to a bus 104. Also connected to the bus 104 area main memory 108 (preferably implemented as random access memory, RAM)and a variety of secondary storage memory 110, such as a hard drive 112and a removable storage medium 114. The removable medium storage device114 may represent, for example, a floppy disk drive, a CD-ROM drive, amagnetic tape drive, etc. A removable storage medium 116 (such as afloppy disk, a compact disk, a magnetic tape, etc.) containing controllogic and/or data recorded therein may be inserted into the removablemedium storage medium 114. The computer system 102 includes appropriatesoftware for reading the control logic and/or the data from theremovable medium storage device 114 once inserted in the removablemedium storage device 114. A monitor 120 can be used as connected to thebus 104 to visualize the structure determination data.

[0148] Amino acid, encoding nucleotide or other sequence and/or x-raydiffraction data of the present invention may be stored in a well knownmanner in the main memory 108, any of the secondary storage devices 110,and/or a removable storage device 116. Software for accessing andprocessing the amino acid sequence and/or x-ray diffraction data (suchas search tools, comparing tools, etc.) reside in main memory 108 duringexecution.

[0149] Three Dimensional Structure Determination One or more computermodeling steps and/or computer algorythms are used to provide amolecular 3-D model of a cleaved PNS SCP, using amino acid sequence datafrom FIGS. 1, 8, 10 or 11 (or variants thereof) and/or x-ray diffractiondata. If only the amino acid sequence is used, for three-dimensionalstructure determination then a suitable modeling program can be used,e.g., LINUS (Rose et. al. Proteins: Structure, Function and Genetics(June, 1995) and references cited herein. It is preferred that the PNSSCP model has no or A1a-substituted (for surface) residues in disallowedregions of the Ramachandran plot, and gives a positive 3D-1D profile(Luthy et al., Nature 356:83-85 (1992)), suggesting that all theresidues are in acceptable environments (Kraulis (1991), infra).Alternatively, the dissallowed regions can be corrected by the use ofsuitable algorythms, such as the RAVE program described herein. Phasedetermination is optionally used for solving the three-dimensionalstructure of a cleaved PNS SCP. This structure can then be used for RDDof modulators of PNS SCP neuraminidase, endothelin cathepsin A or otherbiological activity, e.g., which is relevant to a PNS SCP relatedpathology.

[0150] Density Modification and Map Interpretation. Electron densitymaps can be calculated using such programs as those from the CCP4computing package (SERC (UK) Collaborative Computing Project 4,Daresbury Laboratory, UK, 1979). Cycles of two-fold averaging canfurther be used, such as with the program RAVE (Kleywegt & Jones, Baileyet al., eds., First Map to Final Model, SERC Daresbury Laboratory, UK,pp.59-66 (1994)) and gradual model expansion. For map visualization andmodel building a program usch as “O” (Jones (1991), infra) can be used.

[0151] Refinement and Model Validation. Rigid body and positionalrefinement can be carried out using a program such as X-PLOR (Brünger(1992), infra), e.g., with the stereochemical parameters of Engh andHuber (Acta Cryst. A47:392-400 (1991)). If the model at this stage inthe averaged maps still misses residues (e.g., at least 5-10 persubunit), the some or all of the missing residues can be incorporated inthe model during additional cycles of positional refinement and modelbuilding. The refinement procedure can start using data from lowerresolution (e.g., 25-10 Å to 10-3.0 Å and then gradually extended toinclude data from 12-6 Å to 3.0-1.5 Å). β-values for individual atomscan be refined once data between 2.9 and 1.5 Å has been added.Subsequently waters can be gradually added. A program such as ARP(Lamzin and Wilson, Acta Cryst. D49:129-147 (1993)) can be used to addcrystallographic waters and as a tool to check for bad areas in themodel. Programs such as PROCHECK (Lackowski et al., J. Appl. Cryst.26:283-291 (1993)), WHATIF (Vriend, J. Mol. Graph. 8:52-56 (1990)) andPROFILE 3D (Lüthy et al., Nature 356:83-85 (1992)), as well as thegeometrical analysis generated by X-PLOR can be been used to check thestructure for errors. For the final refinement cycle; 20-5% of theweakest data can be rejected using a IF_(obs)I/σ cutoff and anisotropicscaling between F_(obs) and F_(calc) applied after careful assessment ofthe quality and completeness of the data

[0152] Structure Analysis. A program such as DSSP can be used to assignthe secondary structure elements (Kabsch and Sander (1983), infra). Aprogram such as SUPPOS (from the BIOMOL crystallographic computingpackage) can be used to for some or all of the least-squaressuperpositions of various models and parts of models. Solvent accessiblesurfaces and electrostatic potentials can be calculated using suchprograms as GRASP (Nicholls et al. (1991), infra).

[0153] Structure Determination. The structure of a PNS SCP can thus besolved with the molecular replacement procedure such as by using X-PLOR(Brünger (1992), infra). A partial search model for the monomer can beconstructed using a related protein, such as wheat serinecarboxypeptidase structure (Liao et al. (1992), infra). The rotation andtranslation function can be used to yield two or more orientations andpositions for two subunits to form a physiological dimer as determinedbased on their interactions. Cyclical two-fold density averaging canalso be done using the RAVE program and model expansion can also be usedto add missing residues for each monomer, resulting in a model with95-99.9% of the total number residues. The model can be refined in aprogram such as X-PLOR (Brünger (1992), supra), to a suitablecrystallographic R_(factor). The model data is then saved on computerreadable medium for use in further analysis, such as rational drugdesign.

[0154] Rational Design of Drugs that Interact with the PNS SCP. Thedetermination of the three dimensional structure of a cleaved PNS SCP,as described herein, provides a basis for the design of new and specificligands for the diagnosis and/or treatment of at least one PNSSCP-related pathology. Several approaches can be taken for the use ofthe crystal structure of a PNS SCP in the rational design of ligands ofthis protein. A computer-assisted, manual examination of the active sitestructure is optionally done. The use of software such as GRID(Goodford, J. Med. Chem. 28:849-857 (1985)) a program that determinesprobable interaction sites between probes with various functional groupcharacteristics and the enzyme surface—is used to analyze the activesite to determine structures of inhibiting compounds. The programcalculations, with suitable inhibiting groups on molecules (e.g.protonated primary amines) as the probe, are used to identify potentialhotspots around accessible positions at suitable energy contour levels.Suitable ligands, as inhibiting or stimulating modulating compounds orcompositions, are then tested for modulating activities of at least onePNS SCP.

[0155] A diagnostic or therapeutic PNS SCP modulating ligand of thepresent invention can be, but is not limited to, at least one selectedfrom a nucleic acid, a compound, a protein, an element, a lipid, anantibody, a saccharide, an isotope, a carbohydrate, an imaging agent, alipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibodyor fragment thereof, or any combination thereof, which can be detectablylabeled as for labeling antibodies. Such labels include, but are notlimited to, enzymatic labels, radioisotope or radioactive compounds orelements, fluorescent compounds or metals, chemiluminescent compoundsand bioluminescent compounds. Alternatively, any other known diagnosticor therapeutic agent can be used in a method of the invention.

[0156] After preliminary experiments are done to determine the K_(m) ofthe substrate with each enzyme activity of a PNS SCP, the time-dependentnature of modulation of ligand K_(i) values are determined, (e.g., bythe method of Henderson (Biochem. J. 127:321-333 (1972)). For example,the substrate (or blank where appropriate) and enzyme are pre-incubatedin buffer. Reactions are initiated by the addition of substrate.Aliquots are removed over a suitable time course and each quenched byaddition into the aliquots of suitable quenching solution (e.g. sodiumhydroxide in aqueous ethanol). The concentration of product isdetermined, e.g. fluorometrically, using a spectrometer. Plots offluorescence against time can be close to linear over the assay period,and are used to obtain values for the initial velocity in the presence(V_(i)) or absence (V_(o)) of ligand. Error is present in both axes in aHenderson plot, making it inappropriate for standard regression analysis(Leatherbarrow, Trends Biochem. Sci. 15:455-458 (1990)). Therefore,K_(i) values is obtained from the data by fitting to a modified versionof the Henderson equation for competitive inhibition:

Qr ²+(E _(t) −Q−I _(t))r−E _(t)=0

[0157] where (using the notation of Henderson (Biochem. J. 127:321-333(1972)):$Q = {{{K_{i}\left( \frac{A_{t} + K_{a}}{K_{a}} \right)}\quad {and}\quad r} = \frac{V_{o}}{V_{i}}}$

[0158] This equation is solved for the positive root with the constraintthat

Q=K _(i)((A _(t) +K _(a))/K _(a))

[0159] using PROCNLIN from SAS (SAS Institute Inc., Cary, N.C., USA)which performs nonlinear regression using least-square techniques. Theiterative method used is optionally the multivariate secant method,similar to the Gauss-Newton method, except that the derivatives in theTaylor series are estimated from the histogram of iterations rather thansupplied analytically. A suitable convergence criterion is optionallyused, e.g., where there is a change in loss function of less than 10⁻⁸.

[0160] Once modulating ligands are found and isolated or synthesized,crystallographic studies of the compounds complexed to a PNS SCP areperformed. As a non-limiting example, PNS SCP crystals are soaked for 2days in 0.01-100 mM ligand and X-ray diffraction data are collected onan area detector and/or an image plate detector (e.g., a Mar image platedetector) using a rotating anode X-ray source. Data are collected to ashigh a resolution as possible, e.g., 1.5-3.5 Å, and merged with anR-factor on suitable intensities. An atomic model of the inhibitor isbuilt into the difference Fourier map(F_(inhibitor complex)−F_(native)). The model can be refined to asolution in a cycle of simulated annealing (Brünger (1987), infra)involving 10-500 cycles of energy refinement, 100-10,000 1-FS steps ofroom temperature dynamics and/or 10-500 more cycles of energyrefinement. Harmonic restraints are also used for the atom refinement,except for atoms within a 10-15 Å radius of the inhibitor. An R-factoris selected for the model for both the r.m.s. deviations from the idealbond lengths, as well as for the angles, respectively. Directmeasurements of enzyme inhibition provide further confirmation that themodeled ligands are modulators of at least one biological activity of aPNS SC.

[0161] Ligands of a PNS SCP, based on the crystal structure of thisenzyme, are thus also provided by the present invention. Demonstrationof clinically useful levels, e.g., in vivo activity is also important.In evaluating PNS SCP inhibitors for biological activity in animalmodels (e.g., rat, mouse, rabbit) using various oral and parenteralroutes of administration are evaluated Using this approach, it isexpected that modulation of a PNS SCP occurs in suitable animal models,using the ligands discovered by molecular modeling and x-raycrystallography.

[0162] Diagnostic and/or Therapeutic Agents. A diagnostic or therapeuticPNS SCP modulating agent or ligand of the present invention can be, butis not limited to, at least one selected from a nucleic acid, acompound, a protein, an element, a lipid, an antibody, a saccharide, anisotope, a carbohydrate, an imaging agent, a lipoprotein, aglycoprotein, an enzyme, a detectable probe, and antibody or fragmentthereof, or any combination thereof, which can be detectably labeled asfor labeling antibodies, as described herein. Such labels include, butare not limited to, enzymatic labels, radioisotope or radioactivecompounds or elements, fluorescent compounds r metals, chemiluminescentcompounds and bioluminescent compounds. Alternatively, any other knowndiagnostic or therapeutic agent can be used in a method of theinvention.

[0163] A therapeutic agent used in the invention can have a therapeuticeffect on the target cell as a cell or neuron of the peripheral nervoussystem, the effect selected from, but not limited to: correcting adefective gene or protein, a drug action, a toxic effect, a growthstimulating effect, a growth inhibiting effect, a metabolic effect, acatabolic affect, an anabolic effect, a neurohumoral effect, a celldifferentiation stimulatory effect, a cell differentiation inhibitoryeffect, a neuromodulatory effect, a pluripotent stem cell stimulatingeffect, and any other known therapeutic effects that modulates at leastone SC in a cell of the peripheral nervous system can be provided by atherapeutic agent delivered to a target cell via pharmaceuticaladministration or via a delivery vector according to the invention.

[0164] A therapeutic nucleic acid as a therapeutic agent can have, butis not limited to, at least one of the following therapeutic effects ona target cell: inhibiting transcription of a DNA sequence; inhibitingtranslation of an RNA sequence; inhibiting reverse transcription of anRNA or DNA sequence; inhibiting a post-translational modification of aprotein; inducing transcription of a DNA sequence; inducing translationof an RNA sequence; inducing reverse transcription of an RNA or DNAsequence; inducing a post-translational modification of a protein;transcription of the nucleic acid as an RNA; translation of the nucleicacid as a protein or enzyme; and incorporating the nucleic acid into achromosome of a target cell for constitutive or transient expression ofthe therapeutic nucleic acid.

[0165] Therapeutic effects of therapeutic nucleic acids can include, butare not limited to: turning off a defective gene or processing theexpression thereof, such as antisense RNA or DNA; inhibiting viralreplication or synthesis; gene therapy as expressing a heterologousnucleic acid encoding a therapeutic protein or correcting a defectiveprotein; modifying a defective or underexpression of an RNA such as anhnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or anenzyme that generates a compound as a drug or prodrug in pathological ornormal cells expressing the chimeric receptor, and any other knowntherapeutic effects.

[0166] A therapeutic nucleic acid of the invention which encodes, orprovides the therapeutic effect any known toxin, prodrug or gene drugfor delivery to pathogenic nervous cells can also include genes underthe control of a tissue specific transcriptional regulatory sequence(MRSs) specific for pathogenic SC containing cells. Such TRSs wouldfurther limit the expression of the therapeutic agent in the targetcell, according to known methods.

[0167] Non-limiting examples of such PNS SCP modulating agents orligands of the present invention and methods thereof includemethyl/halophenyl-substituted piperizine compounds, such as lidoflazine(see; e.g., Merck Index Monograph 5311 and U.S. Pat. No. 3,267,104, bothentirely incoporated herein by reference). Such compounds were testedand found to inhibit sodium channel activity of at least one PNS SCP ofthe present invention in cell lines expressing at least one PNS SCP,such as PC12, PK1-4 and other isolated or recombinant cells expressingat least one PNS SCP of the present invention. Accordingly, the presentinvention provides PNS SCP modulating agents or ligands asmethyl/halophenyl-substituted piperizines. The substitutions can includealkyl- and/or halophenyl-substituted piperizines.

[0168] Pharmaceutical/Diagnostic Administration. Using PNS SCPmodulating compounds or compositions (including antagonists and agonistsas described above) the present invention further provides a method formodulating the activity of the PNS SCP protein in a cell. In general,agents (antagonists or agonists) which have been identified to inhibitor enhance the activity of PNS SCP can be formulated so that the agentcan be contacted with a cell expressing a PNS SCP protein in vivo. Thecontacting of such a cell with such an agent results in the in vivomodulation of the activity of the PNS SCP proteins. So long as aformulation barrier or toxicity barrier does not exist, agentsidentified in the assays described above will be effective for in vivouse.

[0169] In another embodiment, the invention relates to a method ofadministering PNS SCP or a PNS SCP modulating compound or composition(including PNS SCP antagonists and agonists) to an animal (preferably, amammal (specifically, a human)) in an amount sufficient to effect analtered level of PNS SCP in the animal. The administered PNS SC or PNSSCP modulating compound or composition could specifically effect PNS SCPassociated functions. Further, since PNS SCP is expressed in -peripheralnervous system tissue, administration of PNS SC or PNS SCP modulatingcompound or composition could be used to alter PNS SCP levels in theperipheral nervous system.

[0170] PNS SCP antagonists can be used to treat pain due to trauma orpathology involving the central or peripheral nervous system, orpathologies related to the abnormally high levels of expression of atleast one naturally occurring nervous system specific (NS) sodiumchannel (SC), where a PNS SCP antagonist also inhibits at least one NSSC, or where the pain is mediated to some extent by PN SC. Suchpathologies, include, but are not limited to; inflammatory diseases,neuropathies (e.g., diabetic neuropathy), dystrophies (e.g., reflexsympathetic dystrophy, post-herpetic neuralgia); trauma (tissue damageby any cause); focal pain by any cause.

[0171] Inflammatory diseases can include, but are not limited to,chronic inflammatory pathologies and vascular inflammatory pathologies.Chronic inflammatory pathologies include, but are not limited tosarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, andCrohn's pathology and vascular inflammatory pathologies, such as, butnot limited to, disseminated intravascular coagulation, atherosclerosis,and Kawasaki's pathology.

[0172] PNS SCP agonists can be used to treat pathologies involving thecentral or peripheral nervous system, or pathologies related to theabnormally low levels of expression of at least one naturally occurringnervous system specific (NS) sodium channel (SC), where a PNS SCPagonist also enhances or stimulates at least one NS SC. Suchpathologies, include, but are not limited to, neurodegenerativediseases, diseases of the gastrointestinal tract due to dysfunction ofthe enteric nervous system (e.g., colitis, ileitis, inflammatory bowelsyndrome); diseases of the cardiovascular system (e.g., hypertension andcongestive heart failure); diseases of the genitourinary tract involvingsympathetic and parasympathetic innervation (e.g., benign prostratehyperplasia, impotence); diseases of the neuromuscular system (e.g.,muscular dystrophy, multiple sclerosis, epilepsy).

[0173] Neurodegenerative diseases can include, but are not limited to,demyelinating diseases, such as multiple sclerosis and acute transversemyelitis; hyperkinetic movement disorders, such as Huntington's Choreaand senile chorea; hypokinetic movement disorders, such as Parkinson'sdisease; progressive supranucleo palsy; spinocerebellar degenerations,such as spinal ataxia, Friedreich's ataxia; multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph);and systemic disorders Refsum's disease, abetalipoprotemia, ataxia,telangiectasia, and mitochondrial multi-system disorder); demyelinatingcore disorders, such as multiple sclerosis, acute transverse myelitis;disorders of the motor unit, such as neurogenic muscular atrophies(anterior horn cell degeneration, such as amyotrophic lateral sclerosis,infantile spinal muscular atrophy and juvenile spinal muscular atrophy);or any subset thereof.

[0174] Pharmaceutical/diagnostic administration ofdiagnostic/pharmaceutical compound or composition of the invention, fora PNS SC related pathology can be administered by any means that achieveits intended purpose, for example, to treat or prevent a cancer orprecancer us condition.

[0175] The term “protection”, as in “protection from infection ordisease”, as used herein, encompasses “prevention,” “suppression” or“treatment.” “Prevention” involves administration of a Pharmaceuticalcomposition prior to the induction of the disease. “Suppression”involves administration of the composition prior to the clinicalappearance of the disease. “Treatment” involves administration of theprotective composition after the appearance of the disease. It will beunderstood that in human and veterinary medicine, it is not alwayspossible to distinguish between “preventing” and “suppressing” since theultimate inductive event or events can be unknown, latent, or thepatient is not ascertained until well after the occurrence of the eventor events. Therefore, it is common to use the term “prophylaxis” asdistinct from “treatment” to encompass both “preventing” and“suppressing”as defined herein. The term “protection,” as used herein,is meant to include “prophylaxis.” See, e.g., Berker, infra, Goodman,infra, Avery, infra and Katzung, infra, which are entirely incorporatedherein by reference, including all references cited therein. The“protection” provided need not be absolute, i.e., the disease need notbe totally prevented or eradicated, provided that there is astatistically significant improvement relative to a control population.Protection can be limited to mitigating the severity or rapidity ofonset of symptoms of the disease.

[0176] At least one PNS SC modulating compound or composition of theinvention can be administered by any means that achieve the intendedpurpose, using a pharmaceutical composition as previously described.

[0177] For example, administration can be by -various parenteral routessuch as subcutaneous, intravenous, intradernal, intramuscular,intraperitoneal, intranasal, intracranial, transdermal, or buccalroutes. Alternatively, or concurrently, administration can be by theoral route. Parenteral administration can be by bolus injection or bygradual perfusion over time.

[0178] An additional mode of using of a diagnostic/pharmaceuticalcompound or composition of the invention is by topical application. Adiagnostic/pharmaceutical compound or composition of the invention canbe incorporated into topically applied vehicles such as salves orointments.

[0179] For topical applications, it is preferred to administer aneffective amount of a diagnostic/pharmaceutical compound or compositionaccording to the invention to target area, e.g., skin surfaces, mucousmembranes, and the like, which are adjacent to peripheral neurons whichare to be treated. This amount will generally range from about 0.0001 mgto about 1 g of a PNS SC modulating compound per application, dependingupon the area to be treated, whether the use is diagnostic, prophylacticor therapeutic, the severity of the symptoms, and the nature of thetopical vehicle employed. A preferred topical preparation is anointment, wherein about 0.001 to about 50 mg of active ingredient isused per cc of ointment base.

[0180] A typical regimen for treatment or prophylaxis comprisesadministration of an effective amount over a period of one or severaldays, up to and including between one week and about six months.

[0181] It is understood that the dosage of a diagnostic/pharmaceuticalcompound or composition of the invention administered in vivo or invitro will be dependent upon the age, sex, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the diagnostic/pharmaceutical effect desired. Theranges of effective doses provided herein are not intended to belimiting and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one skilled in the relevant arts. See,e.g., Berkow et al., eds., The Merck Manual, 16th edition, Merck andCo., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985); Osol et al., eds.,Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co.,Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology, Appletonand Lange, Norwalk, Conn. (1992), which references are entirelyincorporated herein by reference.

[0182] The total dose required-for each treatment can be administered bymultiple doses or in a single dose. The diagnostic/pharmaceuticalcompound or composition can be administered alone or in conjunction withother diagnostics and/or pharmaceuticals directed to the pathology, ordirected to other symptoms of the pathology.

[0183] Effective amounts of a diagnostic/pharmaceutical compound orcomposition of the invention are from about 0.1 μg to about 100 mg/kgbody weight, administered at intervals of 4-72 hours, for a period of 2hours to 1 year, and/or any range or value therein, such as 0.0001-1.0,1-10, 10-50 and 50-100, 0.0001-0.001, 0.001-0.01, 0.01-1.0, 1.0-10,5-10, 10-20, 20-50 and 50-100 mg/kg, at intervals of 1-4, 4-10, 10-16,16-24, 24-36, 36-48, 48-72 hours, for a period of 1-14, 14-28, or 30-44days, or 1-24 weeks, or any range or value therein.

[0184] The recipients of administration of compounds and/or compositionsof the invention can be any vertebrate animal, such as mammals, birds,bony fish, frogs and toads. Among mammals, the preferred recipients aremammals of the Orders Primata (including humans, apes and monkeys),Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta(including mice, rats, rabbits, and hamsters), and Carnivora (includingcats, and dogs). Among birds, the preferred recipients are turkeys,chickens and other members of the same order. The most preferredrecipients are humans.

[0185] Gene Therapy. A delivery vector of the present invention can be,but is not limited to, a viral vector, a liposome, an anti-PNS SCP oranti-SC antibody, or a SC ligand, one or more of which delivery vectorsis associated with a diagnostic or therapeutic agent.

[0186] The delivery vector can comprise any diagnostic or therapeuticagent which has a therapeutic or diagnostic effect on the target cell.The target cell specificity of the delivery vector is thus provided byuse of a target cell specific delivery vector.

[0187] The delivery vector can also be a recombinant viral vectorcomprising at least one binding domain selected from the groupconsisting of an antibody or fragment, a chimeric binding site antibodyor fragment, a target cell or specific ligand, a receptor which binds atarget cell ligand, an anti-idiotypic antibody, a liposome or othercomponent which is specific for the target cell. A PNS SCP can bealready associated with the target cell, or the delivery vector can bindthe target cell via a ligand to a target cell receptor or vice versa.

[0188] Thus, the therapeutic or diagnostic agent, such as a therapeuticor diagnostic nucleic acid, protein, drug, compound composition and thelike, is delivered preferentially to the target cell, e.g., where thenucleic acid is preferably incorporated into the chromosome of thetarget cell, to the partial or complete exclusion of non-target cells.

[0189] The invention is thus intended to provide delivery vectors,containing one or more therapeutic and/or diagnostic agents, includingvectors suitable for gene therapy.

[0190] In a method of treating a PNS SCP-associated disease in a patientin need of such treatment, functional PNS SCP DNA can be provided to thePNS cells of such patient in a manner and amount that permits theexpression of the PNS SCP protein provided by such gene, for a time andin a quantity sufficient to treat such patient, such as a suitabledelivery vector. Many vector systems are known in die art to providesuch delivery to human patients in need of a gene or protein missingfrom the cell. For example, retrovirus systems can be used, especiallymodified retrovirus systems and especially herpes simplex virus systems.Such methods are provided for, in, for example, the teachings ofBreakefield, et al., The New Biologist 3:203-218 (1991); Huang, Q. etal, Experimental Neurology 115:303-316 (1992), WO93/03743 andWO90/09441. Delivery of a DNA sequence encoding a functional PNS SCPprotein will effectively replace the missing or mutated PNS SCP gene ofthe invention.

[0191] In another embodiment of this invention, the PNS SCP modulatingcompound or composition is expressed as a recombinant gene in a cell, sothat the cells can be transplanted into a mammal, preferably a human inneed of gene therapy. To provide gene therapy to an individual, agenetic sequence which encodes for all or part of the PNS SCP modulatingcompound or composition is added into a vector and introduced into ahost cell. Examples of diseases that can be suitable for gene therapyinclude, but are not limited to, neurodegenerative diseases ordisorders, Alzheimer's, schizophrenia, epilepsy, neoplasms and cancer.Examples of vectors that can be used in gene therapy include, but arenot limited to, defective retroviral, adenoviral, or other viral vectors(Mulligan, R. C., Science 260:926-932 (1993)). See Anderson, GeneTherapy, 246 J. Amer. Med. Assn. 2737 (1980); Friedmann, Progress towardhuman gene therapy, 244 Science 1275 (1989); Anderson, 256 Science 808(1992); human gene therapy protocols published in Human Gene Therapy,Mary Ann Liebert Publishers, N.Y. (1990-1994); Bank et al., 565 Ann.N.Y. Acad. Sci. 37 (1989); LTR-Vectors (U.S. Pat. No. 4,405,712);Ausubel, infra, §§ 9.10-9.17; Jon A. Wolff., ed, Gene Therapeutics:methods and applications of direct gene transfer, Birkhäuser, Boston(1994).

[0192] The means by which the vector carrying the gene can be introducedinto the cell include but is not limited to, microinjection,electroporation, transduction, or transfection using DEAE-Dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art (Sambrook infra, Ausubel, infra).

[0193] Preparations for parenteral administration include sterile oraqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and buffered mediaParenteral vehicles include sodium chloride solution, Ringer's dextroseand sodium chloride, lactated Ringer's, or fixed oils. Intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers, such as those based on Ringer's dextrose, and the like.Preservatives and other additives can also be present, such as, forexample, antimicrobials, antioxidants, chelating agents, inert gases andthe like. See, generally, Osol et al., eds. Remington's PharmaceuticalScience, 16th Ed., (1980).

[0194] In another embodiment, the invention relates to a pharmaceuticalcomposition comprising PNS SC or PNS SCP modulating compound orcomposition in an amount sufficient to alter PNS SCP associatedactivity, and a pharmaceutically acceptable diluent, carrier, orexcipient Appropriate concentrations and dosage unit sizes can bereadily determined by one skilled in the art (See, e.g., Osol et al.ed., Remington's Pharmaceutical Sciences, 16th Ed., Mack, Easton Pa.(1980) and WO 91/19008).

[0195] Included as well in the invention are pharmaceutical compositionscomprising an effective amount of at least one PNS SCP antisenseoligonucleotide, in combination with a pharmaceutically acceptablecarrier. Such antisense oligos include, but are not limited to, at leastone nucleotide sequence of 12-500 bases in length which is complementaryto a DNA sequence of SEQ ID NO:1, or a DNA sequence encoding at least 4amino acids of SEQ ID NO:2 or FIG. 11A-11E.

[0196] Alternatively, the PNS SCP nucleic acid can be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol.

[0197] The PNS SCP gene therapy nucleic acids and the pharmaceuticalcompositions of the invention can be administered by any means thatachieve their intended purpose. For example, administration can be byparenteral, subcutaneous, intravenous, intramuscular, intra-peritoneal,or transdermal routes. The dosage administered will be dependent uponthe age, health, and weight of the recipient kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired.

[0198] Compositions within the scope of this invention include allcompositions wherein the PNS SCP antisense oligonucleotide is containedin an amount effective to achieve enhanced expression of at least onePNS SCP in a peripheral nervous system neuron or ganglion. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is with the skill of the art. Typically, thePNS SCP nucleic acid can be administered to mammals, e.g. humans, at adose of 0.005 to 1 mg/kg/day, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated.

[0199] Suitable formulations for parenteral administration includeaqueous solutions of the PNS SCP nucleic acid in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions can be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension can also contain stabilizers.

[0200] Alternatively, at least one PNS SCP can be coded by DNAconstructs which are administered in the form of virions, which arepreferably incapable of replicating in vivo (see, for example, Taylor,WO 92/06693). For example, such DNA constructs can be administered usingherpes-based viruses (Gage et al., U.S. Pat. No. 5,082,670).Alternatively, PNS SCP antisense RNA sequences, PNS SCP ribozymes, andPNS SCP EGS can be coded by RNA constructs which are administered in theform of virions, such as recombinant, replication deficient retrovirusesor adenoviruses. The preparation of retroviral vectois is well known inthe art (see; for example, Brown et al., “Retroviral Vectors,” in DNACloning: A Practical Approach, Volume 3, IRL Press, Washington, D.C.(1987)).

[0201] Specificity for gene expression in the peripheral nervous systemcan be conferred by using appropriate cell-specific regulatorysequences, such as cell-specific enhancers and promoters. Since proteinphosphorylation is critical for neuronal regulation (Kennedy, “SecondMessengers and Neuronal Function,” in An Introduction to MolecularNeurobiology, Hall, E A, Sinauer Associates, Inc. (1992)), proteinkinase promoter sequences can be used to achieve sufficient levels ofPNS SCP gene expression.

[0202] Thus, gene therapy can be used to alleviate sodium channelrelated pathology by inhibiting the inappropriate expression of aparticular form of PNS SC. Moreover, gene therapy can be used toalleviate such pathologies by providing the appropriate expression levelof a particular form of PNS SCP. In this case, particular PNS SCPnucleic acid sequences can be coded by DNA or RNA constructs which areadministered in the form of viruses, as described above.

[0203] Having now generally described the invention, the same will bemore readily understood through reference to the following Exampleswhich are provided by way of illustration, and are not intended to belimiting of the invention, unless specified.

EXAMPLE 1 Cloning and Sequencing of a PNS SC Encoding Nucleic Acid

[0204] Materials and Methods

[0205] Cell Culture. PC12 cells and PKI-4 PC12 subclones were grown aspreviously described (Mandel et al., 1988). NGF (2.5 S subunit, kindlysupplied by Dr. S. Halegoua, SUNY at Stony Brook), was added to theculture medium at final concentration of 110 ng/ml. The PKI-4 PC12subclone which expresses the cAMP-dependent kinase inhibitor protein(PKI) was also provided by Dr. S. Halegoua (see D'Arcangelo et al., J.Cell Biol. 122:915-921 (1993)).

[0206] PCR Amplification. Total cellular RNA was isolated, according tothe method of Cathala et al. DNA 2:329-335 (1983), from a PC12 subclone(PKI-4) which expresses high levels of the cAM-dependent protein kinaseinhibitor protein. Two μg of total RNA prepared time NGF-treated PKI-4cells was used to synthesize first strand cDNA using random hexamerprimers for the reverse transcriptase reaction. The cDNA then served astemplate for the PCR amplification, using a pair of degenerateoligonucleotide primers that specified a 400 base pair region withinrepeat domain III of the sodium: channel α subunit gene. The 5′ primer(designated YJI:GCGAAGCTT(TC)TIATITT(TC)I(GATC)IAT(ATC)ATGGG (SEQ IDNO:3), underline indicates a HindIII restriction site), corresponded toamino acids FWLIFSIM at positions 1347-1354 in the type II sodiumchannel gene. The 3′ primer (designated YO1C:GCAGGATCC(AG)TT(AG)AAA(AG)TT(AG)TC(AGT)AT(AGT)AT(AGCT)AC(AGCT)CC (SEQ IDNO:5), underline indicates a BamH1 restriction site) corresponded toamino acids GVIIDNFN at positions 1470-1447 in the type II gene. Theamplification reaction mixture consisted of 5% of the cDNA, 1 mM MgCl₂,0.2 mM dNTPSs, 0.5 μM each primer, Taq polymerase (Perkin-Elmer) in abuffer consisting of 0.1 M KCl, 0.1 M-TRIS HCl (pH 8.3) and gelatin (1mg/ml). The reaction was performed in a Perkin-Elmer thermocycler asfollows: 5 cycles of denaturation (94° C., 1 min.), annealing (37° C., 1min.), and extension (72° C., 1 min) followed by 25 cycles ofdenaturation (94° C., 1 min.), annealing (50° C., 1 min.) and extension(72° C., 1 min.). The PCR products were excised from a low melt agarosegel (SEAPLAQUE GTG, FMC BIOPRODUCTS) and subcloned into a Bluescript IISK plasmid vector previously restricted with HindIII and BamH1. Theclones were screened for cDNA inserts by miniprep (Sambrook et al.,infra) and sequenced in both directions by dideoxy chain termination(Sequenase 2.0 kit, UNITED STATES BIOCHEMICAL). Sequence data wascompiled and analyzed using GENWORKS software (INTELLIGENETICS, INC.,Mountain View, Calif.).

[0207] cDNA Library Construction and Screening. Poly(A)+ mRNA from thePKI-4 PC12 subclone was purified (mRNA purification kit, PHARMACIA) andused to construct a random- and oligo (dT)-primed Lambda ZAP II cDNAlibrary (STRATAGENE CORP., La Jolla, Calif.). The library consisted of5.6×10⁶ independent clones prior to amplification. Screening ofapproximately 4×10⁶ recombinants using the cloned PCR product pPC12-1labeled by random primers (PHARMACIA kit) resulted in isolation of 5cDNAs ranging in size from 1-3 kb. Sequence analysis and comparison topublished sequences established that the two of the cDNAs togetherencoded 3033 bp of the novel sodium channel α subunit, PN1.

[0208] Northern blot analysis and ribonuclease protection assays. Totalcellular RNA was isolated from adult Sprague-Dawley rat brain, spinalcord, superior cervical ganglion, dorsal root ganglion, skeletal muscle,cardiac muscle, and adrenal gland using the standard method of Chirgwin,Biochemistry 18:5294-5299 (1979). RNA was electrophoresed andtransferred to nylon membrane as previously described (Cooperman et al,Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) (DURALON-UV; STRATAGENECORP.). RNA blots were cross-linked to the nylon using Stratalinker UVcrosslinker (STRATAGENE CORP.) and hybridized to ³²P-UTP-labeledantisense RNA probes generated from the following linearized templates:pPC12-1, pRB211 (Cooperman, infra, 1987), p1B15 (cyclophilin; Danielsonet al., DNA 7:261-267 (1988)), and rat brain type 1, which contains 51bp of intron, 5′ untranslated sequence and 267 bp of coding sequence ofthe type I sodium channel. RNA probes were transcribed with either T3(pPC12-1), T7, (pNach1), or SP6 (pRB211, p1B15) RNA polymerase accordingto the manufacturer's instructions (PROMEGA CORP, Madison, Wis.). Theblots were washed once in 2×SSC, 0.1% NaDodSO₄ for 15 min. at 68° C.,followed by two washes in 0.2×SSC, 0.1% NaDodSO₄ for 15 min. at 68° C.Autoradiography with preflashed XAR-5 film (EASTMAN KODAK CO.,Rochester, N.Y.) was used for quantitation of mRNA by densitometry.

[0209] Ribonuclease protections assays were performed by use of a kit(RPA II, AMBION INC., Austin, Tex.). Total RNA was hybridized with 10⁴cpm of antisense RNA probe generated from pPC12-1. To control fordifferences in the amount of total RNA between samples, we included anantisense RNA probe for β actin, transcribed from pTRI-β-actin (AMBION,INC.).

[0210] In situ hybridization. Tissue preparation and hybridization wereperformed using a modification of the procedure described by Yokouchi etal., Develop. 113:431-444 (1991). SCG and DRG were dissected from adultSprague-Dawley rats and fixed in 4% paraformaldehyde (in 0.1 M PBS) for2-6 hrs. at 4° C. The tissue was then rinsed≈5 min. in 0.1M PBS (pH7.3), cryoprotected in 30% sucrose (in 0.1 M PBS) for 2 hrs. at 4° C.and embedded in O.C.T. (TISSUE-TEK). Cryostat sections (14 μM) werecollected on SUPERFROST/Plus slides (FISHER SCIENTIFIC), dried≈2 hrs. atroom temp., and then stored at −80° C.

[0211] Immediately before prehybridization, sections were brought toroom temp. and rehydrated in 0.1M PBS (pH 7.3) containing 0.3% TritonX-100 for 5 min. Sections were then treated with 0.2 N HCl for 20 min.,washed in 0.1 M PBS for 5 min., and digested with proteinase K (5 μg/mlin 0.1 M PBS) for 40 min. at 37° C. Sections were then postfixed with 4%paraformaldehyde (in 0.1 M PBS), rinsed with 0.1 M PBS containing 0.1 Mglycine for 15 min., and equilibrated in 50% formamide, 2×SSC for 1 hr.(room temp.).

[0212] Sections were hybridized with antisense digoxigenin-labeled RNAprobes transcribed from pPC12-1 or pNach2 (Cooperman et al., Proc. Nat'lAcad. Sci. USA 84:8721 (1987)) according to the manufacturer'sinstructions for RNA labeling with digoxigenin-UTP (BOEHRINGERMANNHEIM). Unlabeled probes were synthesized by replacingdigoxigenin-UTP with rUTP. Each section was covered with ≈100 μl ofhybridization solution containing 20 mM TRIS HCl (pH 8.0), 2.5 mM EDTA,50% formamide, 0.3 M NaCl, 1× Denhardt's, 10% dextran sulfate, 1 mg/mltRNA, and probe at a concentration of 0.7 μg/ml. Sections were thencovered with PARAFILM coverslips and incubated in a humid chamberovernight at 45° C. After hybridization, sections were washed in 50%formamide, 2×SSC at 45° C. for 1 hr., followed by RNase digestion in0.5M NaCl, 10 mM TRIS HCl (pH 8.0), and 20 μg/ml RNase A (BOEHRINGERMANNHEIM). Sections were subsequently washed at 45° C. in 50%formarnide, 2×SSC for 1 hr., and 50% formamide, 1×SSC for 1 hr.

[0213] Immunological detection was performed using a kit (GENIUS 3 KIT,BOEHRINGER MANNHEIM), according to the manufacturer's instructions. Inmost experiments, the sections were incubated in the color solution for≈3-5 hrs. at room temp. Sections were then coverslipped with AQUA-MOUNT(Lerner Laboratories) and stored in the dark.

[0214] Densitometry. Levels of sodium channel mRNA were determined bydensitometric analysis of the autoradiograms using Bio Image software(Millipore Corp., Ann Arbor, Mich.). Levels of RNA were normalized tothe quantitated levels of cyclophilin mRNA.

[0215] Results

[0216] Isolation of a cDNA expressed preferentially in peripheral nerve.D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993) showed previouslythat NGF treatment of PC12 cells increase the level of an ≈11 kb sodiumchannel gene transcript which did not hybridize to probes specific forany of the known sodium channel genes. A transcript identical in sizewas also detected in mRNA from adult rat sympathetic and sensoryganglia, but not in mRNA from brain. These results suggested that thetranscript encoded a new member of the sodium channel gene family(termed Peripheral Nerve type 1 (PN1)).

[0217] To confirm the identity of the PN1 gene, cDNAs from anNGF-treated PC12 subclone which preferentially expresses PN1 mRNA (PKI-4cells) D'Arcangelo et al were amplified by the polymerase chain reaction(PCR), using a pair of degenerate oligonucleotide primers that specify a400 base pair (bp) region of the sodium channel α subunit gene (seeMethods, FIG. 1). Both primers specified putative membrane-spanningregions within repeat domain III, which are highly conserved amongvoltage-gated sodium channels. The amplified regions between the primersinclude the strictly-conserved pore-lining residues, as well as residueswhich are divergent among the different mammalian α subunits. Sequenceanalysis of the PCR products revealed a cDNA, pPC12-1, which encoded aportion of a novel putative sodium channel α subunit (FIG. 1).Additional cDNAs were further isolated which encapsulated the entire PN1coding region.

[0218] To determine whether pPC12-1 encode part of the PN1 gene, thecDNA was used to generate antisense RNA probes for Northern blotanalysis of mRNA from control and NGF-treated PC12 cells (FIG. 2B). Forcomparison, a duplicate blot (FIG. 2A) was hybridized with an antisenseprobe pRB211, which encode a highly-conserved region of the sodiumchannel α subunit (Cooperman et al., Proc. Nat'l Acad. Sci. USA 84:8721(1987)) and which cross-hybridizes with the PN1 transcript, and that, asshown by D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993), levels ofthe detected transcript should increase rapidly and transientlyfollowing NGF treatment (maximal=5 hrs). Comparison of FIGS. 2A and 2Bshows that pPC12-1 fulfilled both of these criteria. Also, consistentwith D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993), we found thatNGF induction of the transcript detected by pPC12-1 is independent ofcAMP-dependent protein kinase activity.

[0219] To isolate additional cDNAs encoding PN1, a random- and oligo(dT)-primed Lambda ZAP II cDNA library (STRATAGENE, 5.6×10⁶ independentclones) was prepared from poly(A)+mRNA isolated from the same PC12subclone from which pPC12-1 was isolated. Screening 4×10⁴ recombinantswith a probe generated from pPC12-1 resulted in isolation of 2additional, overlapping cDNAs which are joined to give a 3033 bp cDNA(FIG. 7). Additional cDNAs were further isolated which encapsulated theentire PN1 coding region.

[0220] Analysis of the deduced primary structure of PN1. As shown inFIG. 8, the deduced primary structure of PN1 encodes repeat domain II ofthe sodium channel α subunit gene. Comparison with the type 11 sodiumchannel shows that the PN1 sequence contains all of the structuralmotifs characteristic of voltage-gated sodium channels, including sixputative transmembrane domains (IIIS1-IIIS6). The S4 domain, thought toserve as the voltage sensor, exhibits the highly-conserved pattern of apositively-charged residue (lysine or arginine) at every third position;Furthermore, the putative pore-lining segments (IIISS1-IIISS2) containresidues shown to be involved in sodium-selective permeation (Heinemannet al., Nature 356:441-443 (1992)) as well as TTX affinity (Terlaue etal., FEBS Lett. 293:93-96 (1991)).

[0221] In addition to such highly-conserved structural features, thesodium channel αsubunit undergoes several characteristicpost-translational modifications. All sodium channels sequenced to dateexhibit a distinctive pattern of asparagine-linked (N-linked)glycosylation sites, which are found almost exclusively in theextracellular loops joining the S5and S6 transmembrane helices. TheN-linked glycosylation sites of PN1 are in good agreement with thispattern; three potential extracellular glycosylation sites are locatedbetween IIIS5 and IIIS6. Two of the sites are also found in the types I,II and III sodium channels.

[0222] The α subunit is phosphorylated by protein kinase C (PKC), anddeduced PN1 sequence contains the highly-conserved consensus PKCphosphorylation-site at serine¹⁵⁰⁶ (FIG. 1). This residue is located inthe cytoplasmic loop joining domains III and IV that has been implicatedin channel inactivation, and mutational analysis has shown that thisserine is required for PKC modulation of channel inactivation (West etal., 1991).

[0223] The entire DNA (FIG. 9A-D) and amino acid (FIG. 10) sequenceswere determined. The rat PN1 amino acid sequence was compared with newhuman sequences (FIG. 11A-E) presented in Example 2.

[0224] In sum, the deduced primary structure of PN1 contains all of thehallmark structural and functional domains characteristics a α subunitthe voltage-gated sodium channel.

[0225] The PN1 gene is expressed preferentially in the PNS. To determinewhether the PN1 gene was expressed preferentially in the PNS, total RNAwas isolated from adult rat brain, spinal cord, SCG, DRG, skeletalmuscle, and cardiac muscle and subjected to Northern blot analysis.Blots were hybridized with the PN1-specific antisense probe generatedfrom pPC12-1. As shown in FIG. 3A, we found high levels of hybridizationto an ≈11 kb transcript in both SCG and DRG. Much lower, but detectablelevels hybridization were seen to transcripts in both spinal cord andbrain. No detectable hybridization was observed to mRNA from skeletalmuscle, cardiac muscle, or liver.

[0226] Ribonuclease (RNase) protection analyses were also prepared.Total RNA was isolated from the same tissues used in Northern blotanalysis, as well as adrenal gland, and hybridized to PN1-specificantisense probe (pPC12-1). mRNA from SCG, DRG, brain, spinal cord, andadrenal gland protected a 343 bp fragment of the PN1 probe (FIG. 4B).The non-protected bases represent oligonucleotide primer and plasmidsequences. The PN1 probe was not protected by mRNA from either skeletalmuscle or cardiac muscle.

[0227] To determine the relative amounts of PN1 mRNA in the varioustissues, autoradiographs from three separate RNase protectionexperiments were analyzed by densitometry. To control for smalldifferences in the amount of total RNA between samples, we included aprobe for a β actin. PN1 mRNA levels in both SCG and DRG areapproximately 40-fold greater than in spinal cord, adrenal gland andbrain.

[0228] The PN1 gene is expressed in sympathetic and sensory neurons. Todetermine whether the PN1 gene is expressed in neurons of peripheralganglia, in situ hybridization was used to examine the cellulardistribution of PN1 mRNA in adult rat SCG and DRG. Cryostat sectionswere hybridized with a PN1 -specific digoxigenin-labeled RNA probe(pPC12-1), which was visualized using an anti-digoxigenin antibodyconjugated to alkaline phosphatase. As shown in FIGS. 4A, B the PN1antisense probe labeled most neuronal cell bodies in both SCG and DRG.To confirm that the hybridization signal was due to binding of the probespecifically to PN mRNA, we performed two different negative controls:(1) Sections were hybridized-with the digoxigenin-labeled probe in thepresence of a 100-fold excess of unlabeled PN1 antisense probe. (2)Previous experiments have shown that SCG and DRG contain extremely lowlevels of type II sodium channel mRNA (Beckh, S., FEBS Lett 262:317-322(1990)). Therefore, we also hybridized sections with a type II-specificantisense probe. As shown, in FIG. 4C-F, both of these controlexperiments greatly reduced the hybridization signal. Also, consistentwith the results of Northern blot and RNase protection analyses, wefound that hybridization of the labeled PN1 probe to sections of adultrat cerebral cortex yielded no detectable staining.

[0229] Although the PN1 probe stained most neuronal cell bodies in bothSCG and DRG, we found that cell-to-cell variability in PN1 mRNA levelsdiffered between the two ganglia. SCG neurons were fairly homogeneous,in that the intensity of reaction product was relatively constantbetween different cells. DRG neurons, however, were quite heterogeneousin that the staining intensity varied considerably from cell to cell.For example, in FIG. 4B, arrows indicate two DRG neurons ofapproximately the same diameter which differ markedly in stainingintensity.

[0230] Finally, we found that the PN2 probe did not stain non-neuronalcells such as satellite cells and Schwann cells. However, it is possiblethat these cells contain very low levels of PN1 mRNA which are notdetectable by this method.

[0231] SCG neurons also express the type I sodium channel gene. EarlierNorthern blot analysis has shown that mRNA from SCG contains twodistinct sodium channel gene transcripts. As we have demonstrated, thelarger, 11 kb transcript encodes the PN1 sodium channel. The smallertranscript, however, has not yet been identified. We hypothesized thatthis smaller transcript encoded the type I sodium channel, becausemoderate levels of type I mRNA have been found in other PNS tissues(Beckh, S., FEBS Lett. 262:317-322 (1990)). To test this hypothesis,Northern blots of SCG mRNA isolated from adult rats were hybridized withan antisense probe specific for the type I sodium channel gene (pNach1,see Methods above). As shown in FIG. 5, the type I-specific probehybridized specifically to the smaller transcript. Furthermore, we havefound that SCG mRNA protects the type I probe in an RNas protectionassay.

[0232] The putative PN1α subunit and type Iα subunit genes aredifferentially regulated during development. Several studies have shownthat the types I, II and III sodium channel genes are differentiallyregulated during development in both the central and peripheral nervoussystems. To determine whether the PN1 and type I genes are alsoindependently regulated during development, we measured their relativemRNA levels in SCG isolated from rats of different postnatal ages. Tovisualize both transcripts simultaneously, Northern blots werehybridized with the conserved sodium channel gene probe pRB211. As shownin FIG. 6A, in SCG removed on postnatal day 7 (P7), the levels of PN1and type I mRNA are approximately equal. However, by P14, their relativeabundance has shifted such that level of PN1 mRNA exceeds that of type Iby ≈*-fold. This increase in ratio of PN1 to type I mRNA levelscontinues for at least the next four postnatal weeks. By P42, PN1 is thepredominant sodium channel gene transcript, with levels of PN1 mRNAseveral-fold greater than that of type I.

[0233] To quantitate the development changes in mRNA levels,autoradiographs from three separate experiments were analyzed bydensitometry. To control for differences in the amount of total RNAbetween lanes, blots were subsequently hybridizing blots with a probefor the internal control cyclophilin. As shown in FIG. 6B, in whichpercent maximum mRNA is plotted versus postnatal age, the shift inrelative abundance of the two transcripts in largely due to adevelopmental decrease in level of type I sodium channel mRNA. From P7to P42, the level of type I mRNA decreases by approximately 80%.

EXAMPLE 2 Drug Screening for PN-1 Antagonists

[0234] The ability of a PNS SCP-ligand (e.g., antagonists and agonists)to inhibit or enhance the activity of a PNS SCP is be evaluated withcells expressing at least one PNS SCP. An assay for PNS SCP activity insuch cells is used to determine the functionality of the PNS SCP proteinin the presence of at least one agent which can act as antagonist oragonist, and thus, agents that interfere or enhance the activity of PNSSCP are identified. Two or more cell lines (each expressing a differentPNS SCP) are used, as well as optionally using one or more cell linesexpressing a CNS specific sodium channel as a control.

[0235] These agents are selected and screened (1) at random; (2) by arational selection; and or (3) by design using for example, computermodeling techniques.

[0236] There are numerous variations of assays which can be used by askilled artisan without the need for undue experimentation in order toisolate, modulating agents or ligands of a PNS SCP. Agent determinationmethods include Computer Assisted Molecular Design (CAMD)) PNS SCP-agentbinding, sophisticated chemical synthesis and testing, targetedscreening, peptide combinatorial library technology, antisensetechnology and/or biological assays, according to known methods. See,e.g., Rapaka et al., eds., Medications Development: Drug Discovery,Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134,NIH Publication No. 93-3638, U.S. Dept. of Health and Human Services,Rockville, Md. (1993); Langone, Methods in Enzymology, Volume 203,Molecular Design and Modeling:Concepts and Applications, Part B,Antibodies and Antigens, Nucleic Acids, Polysaccharides and Drugs,Section III, pp 587-702, Academic Press, New York (1991)).

[0237] Alternatively, cell expression libraries, or other cells are usedto that have been selected or genetically engineered to express anddisplay a PNS SCP via the use of the PNS SCP nucleic acids of theinvention are preferred in such methods, as host cell lines may bechosen which are devoid of related receptors. Rapaka, infra, (1993), atpages 58-65.

[0238] A PNS SCP agent in the context of the present invention refers toany chemical or biological molecule that associates with a PNS SCP invitro, in situ or in vivo, and can be, but is not limited to, synthetic,recombinant or naturally derived chemical compounds and compositions,e.g., organic compounds, nucleic acids, peptides, carbohydrates, vitaminderivatives, hormones, neurotransmitters, viruses or receptor bindingdomains thereof, opsins, rhodopsins, nucleosides, nucleotides,coagulation cascade factors, odorants or pheremones, toxins, growthfactors, platelet activating factors, neuroactive peptides, neurohumors,or any biologically active compound, such as drugs or naturallyoccurring compounds.

[0239] The agents are selected and screened at random or rationallyselected or designed using computer modeling techniques. For randomscreening, potential agents are selected and assayed for their abilityto bind to the PNS SCP, or a fragment thereof. Alternatively, agents maybe rationally selected or designed. As used herein, a agent is said tobe “rationally selected or designed” when the agent is chosen based onthe configuration of at least one specific PNS SCP (e.g., as presentedin FIG. 11). For example, one skilled in the art can readily adaptcurrently available procedures to generate agents capable of binding toa specific peptide sequence in order to generate rationally designedcompounds, such as chemical compounds, nucleic acids or peptides. See,e.g., Rapaka, infra, (1993); Hurby et al., “Application of SyntheticPeptides: Antisense Peptides,” in Synthetic Peptides: A User's Guide,W.H. Freeman, New York (1992), pp. 289-307; and Kaspczak et al.,Biochemistry 28:9230-2938 (1989).

[0240] A method of screening for an agent that modulates the activity ofat least one PNS SCP comprising:

[0241] (a) incubating at least one cell line expressing at least one PNSSCP with an agent to be tested; and

[0242] (b) assaying the at least one cell for the activity of the atleast one PNS SCP protein by measuring the agents effect on PNS SCPbinding or PNS SCP activity preferably the or assay distinguishes theagent's effect on alternative PNS SCP and determines that the agent haslittle or no effect on CNS sodium channels, or has relatively lesseffect on CNS sodium channels.

[0243] Any cell can be used in the above assay so long as it expresses afunctional form of PNS SCP protein and the PNS SCP activity can bemeasured. The preferred expression cells are eukaryotic cells ororganisms. Such cells can be modified to contain DNA sequences encodingthe PNS SCP protein using routine procedures known in the artAlternatively, one skilled in the art can introduce mRNA encoding thePNS SCP protein directly into the cell.

[0244] In an alternative embodiment stem cell populations for eitherneuronal or glial cells can be genetically engineered to express afunctional PNS SCP ion channel. Such cells expressing the PNS SCP ionchannel, can be transplanted to the diseased or injured region of themammal's neurological system (Neural Transplantation. A PracticalApproach, Donnet & Djorklund, eds., Oxford University Press, New York,N.Y. (1992)). In another embodiment, embryonic tissue or fetal neuronscan be genetically engineered to express functional PNS SCP ion channeland transplanted to the diseased or injured region of the mammal'slimbic system. The feasibility of transplanting fetal dopamine neuronsinto Parkinsonian patients has been demonstrated. (Lindvall et al.,Archives of Neurology 46:615-631 (1989)).

[0245] At least two types of approaches are currently used to expressvoltage-dependent sodium channel clones in order to generate functionalchannel proteins. In one approach, mRNA encoding the cloned cDNA isexpressed in Xenopus oocytes. The sodium channel cDNA is cloned into abacterial expression vector such as the pGEM recombinant plasmid(Melton, et al., 1984). Transcription of the cloned cDNA is carried outusing an RNA polymerase such as SP6 polymerase or T7 polymerase with acapping analog such as M⁷G(5′)ppp(5′)G. The resulting RNA (e.g., about50 nl, corresponding to 2-5 ng) is injected into stage V and stage VIoocytes isolated from Xenopus, and incubated for 3-5 days at 19° C.Oocytes axe tested for sodium channel expression with atwo-microelectrode voltage clamp (Trimmer et al, Neuron 3:33-49 1989).

[0246] In an alternative approach, cDNAs encoding a voltage-dependentsodium channel is cloned into any one of a number of mammalianexpression vectors, and transfected into mammalian cells which do notexpress endogenous voltage-dependent sodium channels (such as fibroblastcell lines). Transfected clones are selected expressing the cloned,transfected cDNA. Sodium channel expression is measured with a wholecell voltage clamp technique using a patch electrode (D'Arcangelo etal., J. Cell. Biol. 122:915-921 (1993)).

[0247] Sources of PNS SCPs and Cell Lines Useful for Drug Screening. Anycell line expressing (Naturally, by induction or due to recombinantexpression of a PNS SCP) can be used for drug screening. As anon-limiting example, PC12 cells are mutants deficient in Protein KinaseA (PKA) activity and which express both PN1 and Type II sodium channels.A126-1B2 cells are a cell line which express PN1, but are now discoveredto does not express Type II sodium channels. PKI-4 is a PC12 cell linetransfected with a cDNA encoding a peptide inhibitor of PKA. Each ofthese cell lines can be used as one source of a PNS SCP of the presentinvention, or as a cell line itself to use in drug screening. Treatmentof PC12 cells with NGF reduces both a PNS SCP (PN1) and type II sodiumchannels, while NGF induces only PN1 in A 126-182 cells. PKI-4 cellsexpress a PNS SCP (PN1) without NGF treatment (D'Arcangelo et al, J.Cell Biol. 122:915-921 (1993)).

[0248] Additionally or alternatively, heterologous expression systemscan also be used in which cell lines (such as Chinese Hamster Ovarycells (CHO)) are stably transfected with a cDNA encoding PN-1. Methodsteps for transfecting and stably expressing cDNA to form heterologouscell lines, are well known in the art An advantage of using transfectedcells is that clones are obtained that express very high levels of a PNSSCP, such as PN-1.

[0249] To screen for PNS SCP modulators, as antagonists or agonists,drugs are examined for their ability to:

[0250] (a) inhibit or enhance the binding of radioligands to a PNS SCP(labeled ligand binding reaction), and/or

[0251] (b) to inhibit or enhance ion flux through the channel of the PNSSCP in a cell line that expresses a PNS SCP.

[0252] Labeled ligand binding neurotoxins can be used to characterizePNS sodium channels. For example previous studies have identified atleast six distinct neurotoxin binding sites on previously characterizednon-PNS-sodium channels (reviewed in Lombert et al., FEB 219(2):355-359(1987)). Many of these sites are thought to be allosterically coupled toone another (for review, see Strichartz et al., Ann. Rev. Neurosci.10:237-267 (1987), and references cited therein). In other words,binding of a drug or toxin to a particular neurotoxin site can besensitive to drug binding at not only that site, but other sites on thechannel as well. This is advantageous for a drug screening program inthat for a given labeled ligand, the likelihood of identifying agentsthat preferrentially bind to a PNS SCP is increased.

[0253] The techniques described herein for measuring labeled ligandbinding to a PNS SCP of the invention in intact cells (e.g., PC12 PKI orPNS SCP expressing heterologous cell lines) in suspension are similar tothose described previously for radioligand binding to other sodiumchannels in brain synaptosomal preparations (see, e.g., Catterall et al,J. Biol. Chem. 256(17):8922-8927 (1981)). However, it is well recognizedby those skilled in the art that these techniques are routinely modifiedfor the use of substrate-attached cells or broken cell preparations,based on the teaching and guidance presented herein.

[0254] A126-1B2, PC12, PKI-4 or other cells expressing a PNS SCP cellsare grown using standard techniques, and optionally treated with NGF for1-2 days to induce PN-1 expression. Cells are harvested and tested forion flux activity with alternative potential agents.

[0255] For both radioligands, binding reactions are conducted e.g., at37° C., then stopped. Samples are quickly filtered with vacuum washedwith ice-cold buffer, and bound radioactivity determined byscintillation counting.

[0256] Ion Flux directly tests the ability of a potential PNS SCP agentto inhibit or enhance the activity of a PNS SCP function, by theirability to inhibit or enhance the influx of ion tracers through a PNSSCP.

[0257] Most previous sodium channel studies have employed ²²Na as atracer (for example, see Catterall et al. J. Biol. Chem.256(17):8922-8927 (1981)). However, the high toxicity of ²Na can be adisadvantage for its use in high-throughput drug screening. A less toxicalternative is (¹⁴C) guanidimium ion, influx of which has been shown tobe a reliable indicator of sodium channel opening (Reith, Europ. J.Pharmacol. 188:33-41 (1990)). Accordingly, routine methods can be usedto screen compounds for modulating PNS SCP ion channel activity, e.g.,(¹⁴C) guanidimium ion flux using intact cells expressing at least onePNS SCP. Additionally these methods are well known to be easily modifiedfor use with ²²Na. Similarly, these known method steps could be modifiedfor use with substrate-attached cells or vesicles prepared from brokencells, according to known method steps.

[0258] For a guanidinium flux assay the methods for ²²Na are modifiedfrom those of Reith (Europ. J. Pharmacol. 188:33-41 (1990) for brainsynaptosomes), e.g., as described in Example 2 below. Aliquots of a cellsuspension containing heterologous cells expressing at least one PNS SCPare incubated for 10 minutes at 37° C. in the presence of channelopeners (typically, 100 μM veratridine) and test drugs in a total volumeof 100 μM (0.20-0.25 mg protein). Ion flux is initiated by the additionof HEPES/TRIS solution also containing 4mM guanidine HCl (final) and1000 dpm/nmol (¹⁴C) guanidine. The reaction is continued for 30 secondsand is stopped by the addition of ice-cold incubation buffer, followedby rapid filtration under vacuum over Whatman GF/C filter. The filtersare washed rapidly with ice-cold incubation buffer and radioactivitydetermined by scintillation counting. Nonspecific uptake is determinedin parallel by the inclusion of 1 mM tetrodotoxin during bothpreincubation and uptake.

[0259] Using the guanidinium flux assay several methyl/halophenylsubstituted compounds, such as lidoflazine (see, e.g., Merck IndexMonograph 5311 and U.S. Pat. No. 3,267,104, both entirely incoporatedherein by reference), were tested and found to inhibit sodium channelactivity of at least one PNS SCP of the present invention in cell linesexpressing at least one PNS SCP, with a pIC50 of 6.51 for lidoflazine onPKI-4 cells. Accordingly, the present invention provides PNS SCPmodulating agents as methyl/halophenyl-substituted piperizines.

EXAMPLE 3 Identification of Human PNS SCP Sequence from a HumanPeripheral Nervous System cDNA Library

[0260] Similar to the procedures provided in Example 1, a humanperipheral nervous system cDNA library (as a human DRG library) was usedfor polymerase chain reaction (PCR) amplification. The PCR used a 5′primer corresponding to DNA encoding amino acids 604-611 of SEQ ID NO:2,and a corresponding 3′ primer encoding amino acids 723-731 of SEQ IDNO:2.

[0261] The PCR reaction mixture consisted of 5% of the cDNA, 1 mM MgCl₂,0.2 mM dNTPSs, 0.5 mM, each primer, Taq polymerase (Perkin-Elmer) in abuffer consisting of 0.1 M KCl, 0.1 M TRIS HCl (pH 8.3) and gelatin (1mg/ml). The reaction was performed in a Perkin-Elmer thermocycler asfollows: five cycles of denaturations (94° C., 1 min.), annealing (37°C., 1 min), and extension (72° C., 1 min.), followed by 25 cycles ofdenaturation (94° C., 1 min.), annealing (50° C., 1 min.), and extension(72° C., 1 min.).

[0262] The resulting PCR products provided a human amplified cDNA whichencoded amino acids 646-658 of SEQ ID NO:2, as presented in FIG. 11A-E.

EXAMPLE 4 Cloning and Sequencing of Human PN-1 Sequence from HumanDorsal Root Ganglion cDNA Library

[0263] As in Examples 1 and 3 above, additional PCR primerscorresponding to SEQ ID NO:1 are used to isolate clones from the humanDRG cDNA library which encompass the entire coding region of one or morehuman PNS SCPs of the present invention. A 5′ primer includes thesequence 5′TTTGTGCCCCACAGACCCCAG3′ (SEQ ID NO: 13) and a 3′ primerincludes the sequence 5′ ACACAAATTCTTGATCTGGAATTGCT3′(SEQ ID NO: 14) or5′CAACCTC AGACAGAGAG CAATGA 3′ (SEQ ID NO: 15), which are used fornested PCR. According to Examples 1 and 3 above, PCR is performed toobtain cDNAs encoding a human PNS SCP.

[0264] Additional PCR is performed by “walking” 5′ or 3′ of the sequencecorresponding to the above PCR product. In this way cDNAs encompassingthe entire coding region of one or more human PNS SCPs are provided.

[0265] The resulting additional cDNA clones or PCR products, encodingthe entire human PNS SCP, are subcloned into a plasmid vector previouslyrestricted with suitable restriction sites. The clones are screened forcDNA inserts by miniprep (Sambrook et al., infra) and sequenced in bothdirections by dideoxy chain termination (Sequenase 2.0 kit, UnitedStates Biochemical). Sequence data is compiled and analyzed usingGeneWorks software (IntelliGenetics, Inc., Mountain View, Calif.). Theexpected alternative amino acid sequences for a human PN1 sequence orpresented in FIG. 11A-D and as SEQ ID NOS:7, 11 and 12, where Xaarepresents 0, 1, 2 or 3 amino acids.

[0266] Transcripts of the size of the resulting human PNS SCP are thenconfirmed to be present in human PNS mRNA or cDNA (encoding a 1970-1990amino acid sequence of FIG. 11A-E). However, as in Example 1, suchtranscripts are not expected to be detected in mRNA from brain. Thisexpected result confirms new human members of the sodium channel genefamily (termed Human Peripheral Nerve type 1 (HUMPN1A (deposited as ATCCNo. ______) and HUMPN1B (Deposited as ATCC No. ______) of FIG. 11A-E,where X is 0, 1, 2 or 3 of the same or different amino acid).

[0267] Complete DNA and amino acid sequences of novel human PN1s arethen confirmed and are expected to contain all of the structural andfunctional domain characteristics of an α subunit of a mammalianvoltage-gated sodium channel.

[0268] All references cited herein, including journal articles orabstracts, published or corresponding U.S. or foreign patentapplications, issued U.S. or foreign patents, or any other references,are entirely incorporated by reference herein, including all data,tables, figures, and text presented in the cited references. Theforegoing description of the specific embodiments will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the invention. Therefore,such adaptations and modifications are intended to be within the meaningand range of equivalents of the disclosed embodiments, based on theteaching and guidance presented herein. It is to be understood that thephraseology or terminology herein is for the purpose of description andnot of limitation, such that the terminology or phraseology f thepresent specification is to be interpreted by the skilled artisan inlight of the teachings and guidance presented herein, in combinationwith the knowledge of one of ordinary skill in the art.

1 19 3033 base pairs nucleic acid both both DNA (genomic) CDS 1..3033 1AGG AAC CTT GTG GTC CTG AAC CTG TTT CTG GCT CTT TTG CTG AGT TCC 48 ArgAsn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser 1 5 10 15TTT AGT TCT GAC AAT CTT ACA GCA ATT GAG GAA GAC ACC GAT GCA AAC 96 PheSer Ser Asp Asn Leu Thr Ala Ile Glu Glu Asp Thr Asp Ala Asn 20 25 30 AACCTC CAG ATC GCA GTG GCC AGA ATT AAG AGG GGA ATC AAT TAC GTG 144 Asn LeuGln Ile Ala Val Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val 35 40 45 AAA CAGACC CTG CGT GAA TTC ATT CTA AAA TCA TTT TCC AAA AAG CCA 192 Lys Gln ThrLeu Arg Glu Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro 50 55 60 AAG GGC TCCAAG GAC ACA AAA CGA ACA GCA GAT CCC AAC AAC AAG AAA 240 Lys Gly Ser LysAsp Thr Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys 65 70 75 80 GAA AAC TATATT TCA AAC CGT ACC CTT GCG GAG ATG AGC AAG GAT CAC 288 Glu Asn Tyr IleSer Asn Arg Thr Leu Ala Glu Met Ser Lys Asp His 85 90 95 AAT TTC CTC AAAGAA AAG GAT AGG ATC AGT GGT TAT GGC AGC AGT CTA 336 Asn Phe Leu Lys GluLys Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu 100 105 110 GAC AAA AGC TTTATG GAT GAA AAT GAT TAC CAG TCC TTT ATC CAT AAC 384 Asp Lys Ser Phe MetAsp Glu Asn Asp Tyr Gln Ser Phe Ile His Asn 115 120 125 CCC AGC CTC ACAGTG ACA GTG CCA ATT GCA CCT GGG GAG TCT GAT TTG 432 Pro Ser Leu Thr ValThr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu 130 135 140 GAG ATT ATG AACACA GAA GAG CTT AGC AGT GAC TCA GAC AGT GAC TAC 480 Glu Ile Met Asn ThrGlu Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr 145 150 155 160 AGC AAA GAGAAA CGG AAC CGA TCA AGC TCT TCT GAG TGC AGC ACT GTT 528 Ser Lys Glu LysArg Asn Arg Ser Ser Ser Ser Glu Cys Ser Thr Val 165 170 175 GAC AAC CCTCTG CCA GGA GAA GAG GAG GCT GAA GCA GAG CCC GTA AAC 576 Asp Asn Pro LeuPro Gly Glu Glu Glu Ala Glu Ala Glu Pro Val Asn 180 185 190 GCA GAT GAGCCT GAA GCC TGC TTT ACA GAT GGT TGT GTG AGG AGA TTT 624 Ala Asp Glu ProGlu Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe 195 200 205 CCA TGC TGCCAA GTT AAT GTA GAC TCT GGG AAA GGG AAA GTT TGG TGG 672 Pro Cys Cys GlnVal Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp 210 215 220 ACC ATC AGGAAG ACG TGC TAC AGG ATA GTT GAA CAC AGC TGG TTT GAA 720 Thr Ile Arg LysThr Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu 225 230 235 240 AGC TTCATC GTT CTC ATG ATC CTG CTC AGC AGT GGA GCT CTG GCT TTT 768 Ser Phe IleVal Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe 245 250 255 GAA GATATC TAT ATT GAA AAG AAA AAG ACC ATT AAG ATT ATC CTG GAG 816 Glu Asp IleTyr Ile Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu 260 265 270 TAT GCTGAC AAG ATA TTC ACC TAC ATC TTC ATT CTG GAA ATG CTT CTA 864 Tyr Ala AspLys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 275 280 285 AAA TGGGTC GCA TAT GGG TAT AAA ACA TAT TTC ACT AAT GCC TGG TGT 912 Lys Trp ValAla Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys 290 295 300 TGG CTGGAC TTC TTA ATT GTT GAT GTG TCT CTA GTT ACT TTA GTA GCC 960 Trp Leu AspPhe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala 305 310 315 320 AACACT CTT GGC TAC TCA GAC CTT GGC CCC ATT AAA TCT CTA CGG ACA 1008 Asn ThrLeu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr 325 330 335 CTGAGG GCC CTA AGA CCC CTA AGA GCC TTG TCT AGA TTT GAA GGA ATG 1056 Leu ArgAla Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met 340 345 350 AGGGTA GTG GTC AAC GCA CTC ATA GGA GCA ATC CCT TCC ATC ATG AAC 1104 Arg ValVal Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn 355 360 365 GTGCTT CTC GTG TGC CTT ATA TTC TGG CTA ATA TTT AGC ATC ATG GGA 1152 Val LeuLeu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly 370 375 380 GTCAAT CTG TTT GCT GGC AAG TTC TAT GAG TGT GTC AAC ACC ACC GAT 1200 Val AsnLeu Phe Ala Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp 385 390 395 400GGG TCA CGA TTT CCT ACA TCT CAA GTT GCA AAC CGT TCT GAG TGT TTT 1248 GlySer Arg Phe Pro Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe 405 410 415GCC CTG ATG AAC GTT AGT GGA AAT GTG CGA TGG AAA AAC CTG AAA GTA 1296 AlaLeu Met Asn Val Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val 420 425 430AAC TTC GAC AAC GTT GGG CTT GGT TAC CTG TCG CTG CTT CAA GTT GCA 1344 AsnPhe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 435 440 445ACA TTC AAG GGC TGG ATG GAT ATT ATG TAT GCA GCA GTT GAC TCT GTT 1392 ThrPhe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val 450 455 460AAT GTA AAT GAA CAG CCG AAA TAC GAA TAC AGT CTC TAC ATG TAC ATT 1440 AsnVal Asn Glu Gln Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile 465 470 475480 TAC TTT GTC ATC TTC ATC ATC TTC GGC TCA TTC TTC ACG TTG AAC CTG 1488Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 485 490495 TTC ATT GGT GTC ATC ATA GAT AAT TTC AAC CAA CAG AAA AAA AAG CTT 1536Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu 500 505510 GGA GGT CAA GAT ATC TTT ATG ACA GAA GAA CAG AAG AAA TAC TAT AAT 1584Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 515 520525 GCA ATG AAG AAG CTT GGG TCC AAA AAA CCA CAA AAA CCA ATT CCA AGG 1632Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg 530 535540 CCA GGG AAC AAA TTC CAA GGA TGT ATA TTT GAC TTA GTG ACA AAC CAA 1680Pro Gly Asn Lys Phe Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln 545 550555 560 GCT TTT GAT ATC ACC ATC ATG GTT CTT ATA TGC CTC AAC ATG GTA ACC1728 Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu Asn Met Val Thr 565570 575 ATG ATG GTA GAA AAA GAG GGG CAA ACT GAG TAC ATG GAT TAT GTT TTA1776 Met Met Val Glu Lys Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu 580585 590 CAC TGG ATC AAC ATG GTC TTC ATT ATC CTG TTC ACT GGG GAG TGT GTG1824 His Trp Ile Asn Met Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val 595600 605 CTG AAG CTA ATC TCC CTC AGA CAT TAC TAC TTC ACT GTG GGT TGG AAC1872 Leu Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn 610615 620 ATT TTG TAT TTT GTG GTA GTG ATC CTC TCC ATT GTA GGA ATG TTT CTC1920 Ile Leu Tyr Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 625630 635 640 GCT GAG ATG ATA GAG AAG TAT TTC GTG TCC CCT ACC CTG TTC CGAGTC 1968 Ala Glu Met Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val645 650 655 ATC CGC CTG GCC AGG ATT GGA CGA ATC CTA CGC CTG ATC AAA GGCGCC 2016 Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala660 665 670 AAG GGG ATC CGC ACT CTG CTC TTT GCT TTG ATG ATG TCC CTT CCTGCG 2064 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala675 680 685 CTG TTC AAC ATC GGC CTC CTG CTT TTC CTG GTC ATG TTC ATC TACGCC 2112 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala690 695 700 ATC TTT GGG ATG TCC AAC TTT GCC TAC GTT AAA AAG GAG GCT GGAATT 2160 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile705 710 715 720 AAT GAC ATG TTC AAC TTT GAG ACT TTT GGC AAC AGC ATG ATCTGC TTG 2208 Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile CysLeu 725 730 735 TTC CAA ATC ACC ACC TCT GCC GGC TGG GAC GGA CTG CTG GCCCCC ATC 2256 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala ProIle 740 745 750 CTC AAC AGC GCA CCT CCC GAC TGT GAC CCT AAA AAA GTT CACCCA GGA 2304 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro Lys Lys Val His ProGly 755 760 765 AGT TCA GTG GAA GGG GAC TGT GGG AAC CCA TCC GTG GGG ATTTTT TAC 2352 Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly Ile PheTyr 770 775 780 TTT GTC AGC TAC ATC ATC ATA TCC TTC CTG GTG GTG GTG AACATG TAC 2400 Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val Val Asn MetTyr 785 790 795 800 ATC GCT GTC ATC CTG GAG AAC TTC AGC GTC GCC ACC GAAGAG AGC ACT 2448 Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala Thr Glu GluSer Thr 805 810 815 GAG CCT CTG AGT GAG GAC GAC TTT GAG ATG TTC TAC GAGGTC TGG GAG 2496 Glu Pro Leu Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu ValTrp Glu 820 825 830 AAG TTC GAC CCT GAC GCC ACT CAG TTC ATA GAG TTC TGCAAG CTC TCT 2544 Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Phe Cys LysLeu Ser 835 840 845 GAC TTT GCA GCT GCC CTG GAT CCT CCC CTC CTC ATC GCAAAG CCA AAC 2592 Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Leu Ile Ala LysPro Asn 850 855 860 AAA GTC CAG CTC ATT GCC ATG GAC CTG CCC ATG GTG AGTGGA GAC CGC 2640 Lys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser GlyAsp Arg 865 870 875 880 ATC CAC TGC CTG GAC ATC TTG TTT GCT TTT ACA AAGCGG GTC CTG GGT 2688 Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys ArgVal Leu Gly 885 890 895 GAG GGT GGA GAG ATG GAT TCT CTT CGT TCA CAG ATGGAA GAA AGG TTC 2736 Glu Gly Gly Glu Met Asp Ser Leu Arg Ser Gln Met GluGlu Arg Phe 900 905 910 ATG TCA GCC AAT CCT TCT AAA GTG TCC TAT GAA CCCATC ACG ACC ACA 2784 Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro IleThr Thr Thr 915 920 925 CTG AAG AGA AAA CAA GAG GAG GTG TCC GCG ACT ATCATT CAG CGT GCT 2832 Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Thr Ile IleGln Arg Ala 930 935 940 TAC AGA CGG TAT CGC CTC AGA CAA CAC GTC AAG AATATA TCG AGT ATA 2880 Tyr Arg Arg Tyr Arg Leu Arg Gln His Val Lys Asn IleSer Ser Ile 945 950 955 960 TAC ATA AAA GAT GGA GAC AGG GAT GAT GAT TTGCCC AAT AAA GAA GAT 2928 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu ProAsn Lys Glu Asp 965 970 975 ACA GTT TTT GAT AAC GTG AAC GAG AAC TCA AGTCCG GAA AAG ACA GAT 2976 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser ProGlu Lys Thr Asp 980 985 990 GTA ACT GCC TCA ACC ATC TCG CCA CCT TCC TATGAC AGT GTC ACA AAG 3024 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser Tyr AspSer Val Thr Lys 995 1000 1005 CCA GAT CAA 3033 Pro Asp Gln 1010 1011amino acids amino acid linear protein 2 Arg Asn Leu Val Val Leu Asn LeuPhe Leu Ala Leu Leu Leu Ser Ser 1 5 10 15 Phe Ser Ser Asp Asn Leu ThrAla Ile Glu Glu Asp Thr Asp Ala Asn 20 25 30 Asn Leu Gln Ile Ala Val AlaArg Ile Lys Arg Gly Ile Asn Tyr Val 35 40 45 Lys Gln Thr Leu Arg Glu PheIle Leu Lys Ser Phe Ser Lys Lys Pro 50 55 60 Lys Gly Ser Lys Asp Thr LysArg Thr Ala Asp Pro Asn Asn Lys Lys 65 70 75 80 Glu Asn Tyr Ile Ser AsnArg Thr Leu Ala Glu Met Ser Lys Asp His 85 90 95 Asn Phe Leu Lys Glu LysAsp Arg Ile Ser Gly Tyr Gly Ser Ser Leu 100 105 110 Asp Lys Ser Phe MetAsp Glu Asn Asp Tyr Gln Ser Phe Ile His Asn 115 120 125 Pro Ser Leu ThrVal Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu 130 135 140 Glu Ile MetAsn Thr Glu Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr 145 150 155 160 SerLys Glu Lys Arg Asn Arg Ser Ser Ser Ser Glu Cys Ser Thr Val 165 170 175Asp Asn Pro Leu Pro Gly Glu Glu Glu Ala Glu Ala Glu Pro Val Asn 180 185190 Ala Asp Glu Pro Glu Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe 195200 205 Pro Cys Cys Gln Val Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp210 215 220 Thr Ile Arg Lys Thr Cys Tyr Arg Ile Val Glu His Ser Trp PheGlu 225 230 235 240 Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly AlaLeu Ala Phe 245 250 255 Glu Asp Ile Tyr Ile Glu Lys Lys Lys Thr Ile LysIle Ile Leu Glu 260 265 270 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe IleLeu Glu Met Leu Leu 275 280 285 Lys Trp Val Ala Tyr Gly Tyr Lys Thr TyrPhe Thr Asn Ala Trp Cys 290 295 300 Trp Leu Asp Phe Leu Ile Val Asp ValSer Leu Val Thr Leu Val Ala 305 310 315 320 Asn Thr Leu Gly Tyr Ser AspLeu Gly Pro Ile Lys Ser Leu Arg Thr 325 330 335 Leu Arg Ala Leu Arg ProLeu Arg Ala Leu Ser Arg Phe Glu Gly Met 340 345 350 Arg Val Val Val AsnAla Leu Ile Gly Ala Ile Pro Ser Ile Met Asn 355 360 365 Val Leu Leu ValCys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly 370 375 380 Val Asn LeuPhe Ala Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp 385 390 395 400 GlySer Arg Phe Pro Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe 405 410 415Ala Leu Met Asn Val Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val 420 425430 Asn Phe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 435440 445 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val450 455 460 Asn Val Asn Glu Gln Pro Lys Tyr Glu Tyr Ser Leu Tyr Met TyrIle 465 470 475 480 Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe ThrLeu Asn Leu 485 490 495 Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln GlnLys Lys Lys Leu 500 505 510 Gly Gly Gln Asp Ile Phe Met Thr Glu Glu GlnLys Lys Tyr Tyr Asn 515 520 525 Ala Met Lys Lys Leu Gly Ser Lys Lys ProGln Lys Pro Ile Pro Arg 530 535 540 Pro Gly Asn Lys Phe Gln Gly Cys IlePhe Asp Leu Val Thr Asn Gln 545 550 555 560 Ala Phe Asp Ile Thr Ile MetVal Leu Ile Cys Leu Asn Met Val Thr 565 570 575 Met Met Val Glu Lys GluGly Gln Thr Glu Tyr Met Asp Tyr Val Leu 580 585 590 His Trp Ile Asn MetVal Phe Ile Ile Leu Phe Thr Gly Glu Cys Val 595 600 605 Leu Lys Leu IleSer Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn 610 615 620 Ile Leu TyrPhe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 625 630 635 640 AlaGlu Met Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val 645 650 655Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 660 665670 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 675680 685 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala690 695 700 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala GlyIle 705 710 715 720 Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser MetIle Cys Leu 725 730 735 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly LeuLeu Ala Pro Ile 740 745 750 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro LysLys Val His Pro Gly 755 760 765 Ser Ser Val Glu Gly Asp Cys Gly Asn ProSer Val Gly Ile Phe Tyr 770 775 780 Phe Val Ser Tyr Ile Ile Ile Ser PheLeu Val Val Val Asn Met Tyr 785 790 795 800 Ile Ala Val Ile Leu Glu AsnPhe Ser Val Ala Thr Glu Glu Ser Thr 805 810 815 Glu Pro Leu Ser Glu AspAsp Phe Glu Met Phe Tyr Glu Val Trp Glu 820 825 830 Lys Phe Asp Pro AspAla Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser 835 840 845 Asp Phe Ala AlaAla Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn 850 855 860 Lys Val GlnLeu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg 865 870 875 880 IleHis Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly 885 890 895Glu Gly Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe 900 905910 Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr 915920 925 Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala930 935 940 Tyr Arg Arg Tyr Arg Leu Arg Gln His Val Lys Asn Ile Ser SerIle 945 950 955 960 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu Pro AsnLys Glu Asp 965 970 975 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser ProGlu Lys Thr Asp 980 985 990 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser TyrAsp Ser Val Thr Lys 995 1000 1005 Pro Asp Gln 1010 29 base pairs nucleicacid single linear cDNA misc_feature 12 /note= “Base is Inosine”misc_feature 15 /note= “Base is Inosine” misc_feature 19 /note= “Base isInosine” misc_feature 21 /note= “Base is Inosine” 3 GCGAAGCTTYTNATNTTYNN NATHATGGG 29 8 amino acids amino acid single linear peptide 4Phe Trp Leu Ile Phe Ser Ile Met 1 5 34 base pairs nucleic acid singlelinear cDNA 5 GCAGGATCCR TTRAAARTTR TCDATDATNA CNCC 34 8 amino acidsamino acid single linear peptide 6 Gly Val Ile Ile Asp Asn Phe Asn 1 52005 amino acids amino acid single linear peptide 7 Met Ala Arg Ser ValLeu Val Pro Pro Gly Pro Asp Ser Phe Arg Phe 1 5 10 15 Phe Thr Arg GluSer Leu Ala Ala Ile Glu Gln Arg Ile Ala Glu Glu 20 25 30 Lys Ala Lys ArgPro Lys Gln Glu Arg Lys Asp Glu Asp Asp Glu Asn 35 40 45 Gly Pro Lys ProAsn Ser Asp Leu Glu Ala Gly Lys Ser Leu Pro Phe 50 55 60 Ile Tyr Gly AspIle Pro Pro Glu Met Val Ser Glu Pro Leu Glu Asp 65 70 75 80 Leu Asp ProTyr Tyr Ile Asn Lys Lys Thr Phe Ile Val Leu Asn Lys 85 90 95 Gly Lys AlaIle Ser Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu 100 105 110 Thr ProPhe Asn Pro Ile Arg Lys Leu Ala Ile Lys Ile Leu Val His 115 120 125 SerLeu Phe Asn Val Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Val 130 135 140Phe Met Thr Met Ser Asn Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr 145 150155 160 Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Leu Ala165 170 175 Arg Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asn Pro TrpAsn 180 185 190 Trp Leu Asp Phe Thr Val Ile Thr Phe Ala Tyr Val Thr GluPhe Val 195 200 205 Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg ValLeu Arg Ala 210 215 220 Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys ThrIle Val Gly Ala 225 230 235 240 Leu Ile Gln Ser Val Lys Lys Leu Ser AspVal Met Ile Leu Thr Val 245 250 255 Phe Cys Leu Ser Val Phe Ala Leu IleGly Leu Gln Leu Phe Met Gly 260 265 270 Asn Leu Arg Asn Lys Cys Leu GlnTrp Pro Pro Asp Asn Ser Thr Phe 275 280 285 Glu Ile Asn Ile Thr Ser PhePhe Asn Asn Ser Leu Asp Trp Asn Gly 290 295 300 Thr Ala Phe Asn Arg ThrVal Asn Met Phe Asn Trp Asp Glu Tyr Ile 305 310 315 320 Glu Asp Lys SerHis Phe Tyr Phe Leu Glu Gly Gln Asn Asp Ala Leu 325 330 335 Leu Cys GlyAsn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly Tyr Ile 340 345 350 Cys ValLys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp 355 360 365 ThrPhe Ser Trp Ala Phe Leu Ser Leu Phe Arg Leu Met Thr Gln Asp 370 375 380Phe Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr 385 390395 400 Tyr Met Ile Phe Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu405 410 415 Ile Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu GlnAsn 420 425 430 Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu PheGln Gln 435 440 445 Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala GlnAla Ala Ala 450 455 460 Ala Ala Ala Ser Ala Glu Ser Arg Asp Phe Ser GlyAla Gly Gly Ile 465 470 475 480 Gly Val Phe Ser Glu Ser Ser Ser Val AlaSer Lys Leu Ser Ser Lys 485 490 495 Ser Glu Lys Glu Leu Lys Asn Arg ArgLys Lys Lys Lys Gln Lys Glu 500 505 510 Gln Ala Gly Glu Glu Glu Lys GluAsp Ala Val Arg Lys Ser Ala Ser 515 520 525 Glu Asp Ser Ile Arg Lys LysGly Phe Gln Phe Ser Leu Glu Gly Ser 530 535 540 Arg Leu Thr Tyr Glu LysArg Phe Ser Ser Pro His Gln Ser Leu Leu 545 550 555 560 Ser Ile Arg GlySer Leu Phe Ser Pro Arg Arg Asn Ser Arg Ala Ser 565 570 575 Leu Phe AsnPhe Lys Gly Arg Val Lys Asp Ile Gly Ser Glu Asn Asp 580 585 590 Phe AlaAsp Asp Glu His Ser Thr Phe Glu Asp Asn Asp Ser Arg Arg 595 600 605 AspSer Leu Phe Val Pro His Arg His Gly Glu Arg Arg Pro Ser Asn 610 615 620Val Ser Gln Ala Ser Arg Ala Ser Arg Gly Ile Pro Thr Leu Pro Met 625 630635 640 Asn Gly Lys Met His Ser Ala Val Asp Cys Asn Gly Val Val Ser Leu645 650 655 Val Gly Gly Pro Ser Ala Leu Thr Ser Pro Val Gly Gln Leu LeuPro 660 665 670 Glu Gly Thr Thr Thr Glu Thr Glu Ile Arg Lys Arg Arg SerSer Ser 675 680 685 Tyr His Val Ser Met Asp Leu Leu Glu Asp Pro Ser ArgGln Arg Ala 690 695 700 Met Ser Met Ala Ser Ile Leu Thr Asn Thr Met GluGlu Leu Glu Glu 705 710 715 720 Ser Arg Gln Lys Cys Pro Pro Cys Trp TyrLys Phe Ala Asn Met Cys 725 730 735 Leu Ile Trp Asp Cys Cys Lys Pro TrpLeu Lys Val Lys His Val Val 740 745 750 Asn Leu Val Val Met Asp Pro PheVal Asp Leu Ala Ile Thr Ile Cys 755 760 765 Ile Val Leu Asn Thr Leu PheMet Ala Met Glu His Tyr Pro Met Thr 770 775 780 Glu Gln Phe Ser Ser ValLeu Ser Val Gly Asn Leu Val Phe Thr Gly 785 790 795 800 Ile Phe Thr AlaGlu Met Phe Leu Lys Ile Ile Ala Met Asp Pro Tyr 805 810 815 Tyr Tyr PheGln Glu Gly Trp Asn Ile Phe Asp Gly Phe Ile Val Ser 820 825 830 Leu SerLeu Met Glu Leu Gly Leu Ala Asn Val Glu Gly Leu Ser Val 835 840 845 LeuArg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser Trp 850 855 860Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly Ala 865 870875 880 Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe Ala885 890 895 Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys ValCys 900 905 910 Lys Ile Ser Asn Asp Cys Glu Leu Pro Arg Trp His Met HisHis Phe 915 920 925 Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys GlyGlu Trp Ile 930 935 940 Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly GlnThr Met Cys Leu 945 950 955 960 Thr Val Phe Met Met Val Met Val Ile GlyAsn Leu Val Val Leu Asn 965 970 975 Leu Phe Leu Ala Leu Leu Leu Ser SerPhe Ser Ser Asp Asn Leu Ala 980 985 990 Ala Thr Asp Asp Asp Asn Glu MetAsn Asn Leu Gln Ile Ala Val Gly 995 1000 1005 Arg Met Gln Lys Gly IleAsp Phe Val Lys Arg Lys Ile Arg Glu Phe 1010 1015 1020 Ile Gln Lys AlaPhe Val Arg Lys Gln Lys Ala Leu Asp Glu Ile Lys 1025 1030 1035 1040 ProLeu Glu Asp Leu Asn Asn Lys Lys Asp Ser Cys Ile Ser Asn His 1045 10501055 Thr Thr Ile Glu Ile Gly Lys Asp Leu Asn Tyr Leu Lys Asp Gly Asn1060 1065 1070 Gly Thr Thr Ser Gly Ile Gly Ser Ser Val Glu Lys Tyr ValVal Asp 1075 1080 1085 Glu Ser Asp Tyr Met Ser Phe Ile Asn Asn Pro SerLeu Thr Val Thr 1090 1095 1100 Val Pro Ile Ala Leu Gly Glu Ser Asp PheGlu Asn Leu Asn Thr Glu 1105 1110 1115 1120 Glu Phe Ser Ser Glu Ser AspMet Glu Glu Ser Lys Glu Lys Leu Asn 1125 1130 1135 Ala Thr Ser Ser SerGlu Gly Ser Thr Val Asp Ile Gly Ala Pro Ala 1140 1145 1150 Glu Gly GluGln Pro Glu Ala Glu Pro Glu Glu Ser Leu Glu Pro Glu 1155 1160 1165 AlaCys Phe Thr Glu Asp Cys Val Arg Lys Phe Lys Cys Cys Gln Ile 1170 11751180 Ser Ile Glu Glu Gly Lys Gly Lys Leu Trp Trp Asn Leu Arg Lys Thr1185 1190 1195 1200 Cys Tyr Lys Ile Val Glu His Asn Trp Phe Glu Ile PheIle Val Phe 1205 1210 1215 Met Ile Leu Leu Ser Ser Gly Ala Leu Ala PheGlu Asp Ile Tyr Ile 1220 1225 1230 Glu Gln Arg Lys Thr Ile Lys Thr MetLeu Glu Tyr Ala Asp Lys Val 1235 1240 1245 Phe Thr Tyr Ile Phe Ile LeuGlu Met Leu Leu Lys Trp Val Ala Tyr 1250 1255 1260 Gly Phe Gln Met TyrPhe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1265 1270 1275 1280 Ile ValAsp Val Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr 1285 1290 1295Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg 13001305 1310 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val ValAsn 1315 1320 1325 Ala Leu Leu Gly Ala Ile Pro Ser Ile Met Asn Val LeuLeu Val Cys 1330 1335 1340 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met GlyVal Asn Leu Phe Ala 1345 1350 1355 1360 Gly Lys Phe Tyr His Cys Ile AsnTyr Thr Ile Gly Glu Met Phe Asp 1365 1370 1375 Val Ser Val Val Asn AsnTyr Ser Glu Cys Gln Ala Leu Ile Glu Ser 1380 1385 1390 Asn Gln Thr AlaArg Trp Lys Asn Val Lys Val Asn Phe Asp Asn Val 1395 1400 1405 Gly LeuGly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1410 1415 1420Met Asp Ile Met Tyr Ala Ala Val Asp Ser Arg Asn Val Glu Leu Gln 14251430 1435 1440 Pro Lys Tyr Glu Asp Asn Leu Tyr Met Tyr Leu Tyr Phe ValIle Phe 1445 1450 1455 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu PheIle Gly Val Ile 1460 1465 1470 Ile Asp Asn Phe Asn Gln Gln Lys Lys LysPhe Gly Gly Gln Asp Ile 1475 1480 1485 Phe Met Thr Glu Glu Gln Lys LysTyr Tyr Asn Ala Met Lys Lys Leu 1490 1495 1500 Gly Ser Lys Lys Pro GlnLys Pro Ile Pro Arg Pro Ala Asn Lys Phe 1505 1510 1515 1520 Gln Gly MetVal Phe Asp Phe Val Thr Lys Gln Val Phe Asp Ile Ser 1525 1530 1535 IleMet Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr 1540 15451550 Asp Asp Gln Ser Gln Glu Met Thr Asn Ile Leu Tyr Trp Ile Asn Leu1555 1560 1565 Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val Leu Lys LeuIle Ser 1570 1575 1580 Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn IlePhe Asp Phe Val 1585 1590 1595 1600 Val Val Ile Leu Ser Ile Val Gly MetPhe Leu Ala Glu Leu Ile Glu 1605 1610 1615 Lys Tyr Phe Val Ser Pro ThrLeu Phe Arg Val Ile Arg Leu Ala Arg 1620 1625 1630 Ile Gly Arg Ile LeuArg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr 1635 1640 1645 Leu Leu PheAla Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1650 1655 1660 LeuLeu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 1665 16701675 1680 Asn Phe Ala Tyr Val Lys Arg Glu Val Gly Ile Asp Asp Met PheAsn 1685 1690 1695 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe GlnIle Thr Thr 1700 1705 1710 Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro IleLeu Asn Ser Gly Pro 1715 1720 1725 Pro Asp Cys Asp Pro Glu Lys Asp HisPro Gly Ser Ser Val Lys Gly 1730 1735 1740 Asp Cys Gly Asn Pro Ser ValGly Ile Phe Phe Phe Val Ser Tyr Ile 1745 1750 1755 1760 Ile Ile Ser PheLeu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu 1765 1770 1775 Glu AsnPhe Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu 1780 1785 1790Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 17951800 1805 Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala AlaAla 1810 1815 1820 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys ValGln Leu Ile 1825 1830 1835 1840 Ala Met Asp Leu Pro Met Val Ser Gly AspArg Ile His Cys Leu Asp 1845 1850 1855 Ile Leu Phe Ala Phe Thr Lys ArgVal Leu Gly Glu Ser Gly Glu Met 1860 1865 1870 Asp Ala Leu Arg Ile GlnMet Glu Glu Arg Phe Met Ala Ser Asn Pro 1875 1880 1885 Ser Lys Val SerTyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1890 1895 1900 Glu GluVal Ser Ala Ile Val Ile Gln Arg Ala Tyr Arg Arg Tyr Leu 1905 1910 19151920 Leu Lys Gln Lys Val Lys Lys Val Ser Ser Ile Tyr Lys Lys Asp Lys1925 1930 1935 Gly Lys Glu Asp Glu Gly Thr Pro Ile Lys Glu Asp Ile IleThr Asp 1940 1945 1950 Lys Leu Asn Glu Asn Ser Thr Pro Glu Lys Thr AspVal Thr Pro Ser 1955 1960 1965 Thr Thr Ser Pro Pro Ser Tyr Asp Ser ValThr Lys Pro Glu Lys Glu 1970 1975 1980 Lys Phe Glu Lys Asp Lys Ser GluLys Glu Asp Lys Gly Lys Asp Ile 1985 1990 1995 2000 Arg Glu Ser Lys Lys2005 813 amino acids amino acid single linear peptide 8 Asn Leu Val ValLeu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 1 5 10 15 Ser Ser AspAsn Leu Ala Asp Asn Asn Leu Gln Ile Ala Val Arg Gly 20 25 30 Ile Val LysArg Glu Phe Ile Lys Phe Lys Lys Lys Asp Asn Asn Lys 35 40 45 Lys Ile SerAsn Thr Glu Lys Asp Asn Leu Lys Ser Gly Gly Ser Ser 50 55 60 Lys Asp GluAsp Tyr Ser Phe Ile Asn Pro Ser Leu Thr Val Thr Val 65 70 75 80 Pro IleAla Gly Glu Ser Asp Glu Asn Thr Glu Glu Ser Ser Ser Asp 85 90 95 Ser LysGlu Lys Asn Ser Ser Ser Glu Ser Thr Val Asp Pro Glu Glu 100 105 110 GluAla Glu Pro Glu Pro Glu Ala Cys Phe Thr Cys Val Arg Phe Cys 115 120 125Cys Gln Gly Lys Gly Lys Trp Trp Arg Lys Thr Cys Tyr Ile Val Glu 130 135140 His Trp Phe Glu Phe Ile Val Met Ile Leu Leu Ser Ser Gly Ala Leu 145150 155 160 Ala Phe Glu Asp Ile Tyr Ile Glu Lys Thr Ile Lys Leu Glu TyrAla 165 170 175 Asp Lys Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu LysTrp Val 180 185 190 Ala Tyr Gly Tyr Phe Thr Asn Ala Trp Cys Trp Leu AspPhe Leu Ile 195 200 205 Val Asp Val Ser Leu Val Leu Ala Asn Leu Gly TyrSer Leu Gly Ile 210 215 220 Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg ProLeu Arg Ala Leu Ser 225 230 235 240 Arg Phe Glu Gly Met Arg Val Val ValAsn Ala Leu Gly Ala Ile Pro 245 250 255 Ser Ile Met Asn Val Leu Leu ValCys Leu Ile Phe Trp Leu Ile Phe 260 265 270 Ser Ile Met Gly Val Asn LeuPhe Ala Gly Lys Phe Tyr Cys Asn Thr 275 280 285 Gly Phe Ser Val Asn SerGlu Cys Ala Leu Arg Trp Lys Asn Lys Val 290 295 300 Asn Phe Asp Asn ValGly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 305 310 315 320 Thr Phe LysGly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Asn 325 330 335 Val GlnPro Lys Tyr Glu Leu Tyr Met Tyr Tyr Phe Val Ile Phe Ile 340 345 350 IlePhe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile 355 360 365Asp Asn Phe Asn Gln Gln Lys Lys Lys Gly Gly Gln Asp Ile Phe Met 370 375380 Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser 385390 395 400 Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Asn Lys Phe Gln GlyPhe 405 410 415 Asp Val Thr Gln Phe Asp Ile Ile Met Leu Ile Cys Leu AsnMet Val 420 425 430 Thr Met Met Val Glu Gln Met Leu Trp Ile Asn Val PheIle Leu Phe 435 440 445 Thr Gly Glu Cys Val Leu Lys Leu Ile Ser Leu ArgHis Tyr Tyr Phe 450 455 460 Thr Gly Trp Asn Ile Phe Val Val Val Ile LeuSer Ile Val Gly Met 465 470 475 480 Phe Leu Ala Glu Ile Glu Lys Tyr PheVal Ser Pro Thr Leu Phe Arg 485 490 495 Val Ile Arg Leu Ala Arg Ile GlyArg Ile Leu Arg Leu Ile Lys Gly 500 505 510 Ala Lys Gly Ile Arg Thr LeuLeu Phe Ala Leu Met Met Ser Leu Pro 515 520 525 Ala Leu Phe Asn Ile GlyLeu Leu Leu Phe Leu Val Met Phe Ile Tyr 530 535 540 Ala Ile Phe Gly MetSer Asn Phe Ala Tyr Val Lys Glu Gly Ile Asp 545 550 555 560 Met Phe AsnPhe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln 565 570 575 Ile ThrThr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn 580 585 590 SerPro Pro Asp Cys Asp Pro Lys His Pro Gly Ser Ser Val Gly Asp 595 600 605Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Val Ser Tyr Ile Ile Ile 610 615620 Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 625630 635 640 Phe Ser Val Ala Thr Glu Glu Ser Glu Pro Leu Ser Glu Asp AspPhe 645 650 655 Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp AlaThr Gln 660 665 670 Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala Ala AlaLeu Asp Pro 675 680 685 Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln LeuIle Ala Met Asp 690 695 700 Leu Pro Met Val Ser Gly Asp Arg Ile His CysLeu Asp Ile Leu Phe 705 710 715 720 Ala Phe Thr Lys Arg Val Leu Gly GluGly Glu Met Asp Leu Arg Gln 725 730 735 Met Glu Glu Arg Phe Met Asn ProSer Lys Val Ser Tyr Glu Pro Ile 740 745 750 Thr Thr Thr Leu Lys Arg LysGln Glu Glu Val Ser Ala Ile Gln Arg 755 760 765 Ala Tyr Arg Arg Tyr LeuGln Val Lys Ser Ser Ile Tyr Lys Asp Asp 770 775 780 Pro Lys Glu Asp AspAsn Glu Asn Ser Pro Glu Lys Thr Asp Val Thr 785 790 795 800 Ser Thr SerPro Pro Ser Tyr Asp Ser Val Thr Lys Pro 805 810 6452 base pairs nucleicacid double both DNA (genomic) CDS 326..6277 9 GTCGCCTCAT CCTGAGCAGACTGGAAACAG ACTCCGTGCA GGCCTCGCCC GCGCTCCAGT 60 TGCGACTGTA GGGTTTTCATTCCTGCCCAC TGCGCAGACT GGGCTGAGCT AGCCTGGGTA 120 TCCACGATTC GCGACTCGTAGTAACAGGCA CTCTGAGCAA CAGGATTTCA GAGAAAGAAG 180 CAGAGGCAAG AAAGAAGCCTGGGGAGAGAG GAAGACTTTC CTTGGATCAG ACTCCGCAGG 240 TGCACACACC GGGTGGGCATGATCCGTGGG GCCAGGCCTC TTAGGTAAGG AGTCAAAGGG 300 GAAATAAAAC ATACAGGATGAAAAG ATG GCG ATG CTG CCT CCT CCA GGA CCT 352 Met Ala Met Leu Pro ProPro Gly Pro 1015 1020 CAG AGT TTC GTT CAC TTC ACA AAA CAG TCC CTT GCCCTC ATT GAA CAG 400 Gln Ser Phe Val His Phe Thr Lys Gln Ser Leu Ala LeuIle Glu Gln 1025 1030 1035 CGT ATT TCT GAA GAA AAA GCC AAG GAA CAC AAAGAC GAA AAG AAA GAT 448 Arg Ile Ser Glu Glu Lys Ala Lys Glu His Lys AspGlu Lys Lys Asp 1040 1045 1050 GAT GAG GAA GAA GGC CCC AAG CCC AGC AGTGAC TTG GAA GCT GGG AAA 496 Asp Glu Glu Glu Gly Pro Lys Pro Ser Ser AspLeu Glu Ala Gly Lys 1055 1060 1065 CAG CTC CCC TTC ATC TAT GGA GAC ATTCCC CCT GGA ATG GTG TCA GAG 544 Gln Leu Pro Phe Ile Tyr Gly Asp Ile ProPro Gly Met Val Ser Glu 1070 1075 1080 CCC CTG GAG GAC CTG GAC CCA TACTAT GCT GAC AAA AAA ACT TTT ATA 592 Pro Leu Glu Asp Leu Asp Pro Tyr TyrAla Asp Lys Lys Thr Phe Ile 1085 1090 1095 1100 GTA TTG AAC AAA GGG AAAGCA ATC TTC CGT TTC AAC GCC ACC CCT GCT 640 Val Leu Asn Lys Gly Lys AlaIle Phe Arg Phe Asn Ala Thr Pro Ala 1105 1110 1115 TTG TAC ATG CTG TCTCCC TTC AGT CCT CTA AGA AGA ATA TCT ATT AAG 688 Leu Tyr Met Leu Ser ProPhe Ser Pro Leu Arg Arg Ile Ser Ile Lys 1120 1125 1130 ATC TTA GTG CACTCC TTA TTC AGC ATG CTA ATC ATG TGC ACA ATT CTG 736 Ile Leu Val His SerLeu Phe Ser Met Leu Ile Met Cys Thr Ile Leu 1135 1140 1145 ACG AAC TGCATA TTC ATG ACC TTG AGC AAC CCT CCA GAA TGG ACC AAA 784 Thr Asn Cys IlePhe Met Thr Leu Ser Asn Pro Pro Glu Trp Thr Lys 1150 1155 1160 AAT GTAGAG TAC ACT TTT ACT GGG ATA TAT ACT TTT GAA TCA CTC ATA 832 Asn Val GluTyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile 1165 1170 1175 1180AAA ATC CTT GCA AGA GGC TTT TGC GTG GGA GAA TTC ACC TTC CTC CGT 880 LysIle Leu Ala Arg Gly Phe Cys Val Gly Glu Phe Thr Phe Leu Arg 1185 11901195 GAC CCT TGG AAC TGG CTG GAC TTT GTT GTC ATT GTT TTT GCG TAT TTA 928Asp Pro Trp Asn Trp Leu Asp Phe Val Val Ile Val Phe Ala Tyr Leu 12001205 1210 ACA GAA TTT GTA AAC CTA GGC AAT GTT TCA GCT CTT CGA ACT TTCAGA 976 Thr Glu Phe Val Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg1215 1220 1225 GTC TTG AGA GCT TTG AAA ACT ATT TCT GTA ATC CCA GGA CTAAAG ACC 1024 Val Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu LysThr 1230 1235 1240 ATC GTG GGG GCC CTG ATC CAG TCA GTG AAG AAG CTC TCTGAC GTC ATG 1072 Ile Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser AspVal Met 1245 1250 1255 1260 ATC CTC ACT GTG TTC TGT CTC AGT GTG TTT GCACTA ATT GGA CTA CAG 1120 Ile Leu Thr Val Phe Cys Leu Ser Val Phe Ala LeuIle Gly Leu Gln 1265 1270 1275 CTG TTT ATG GGC AAC TTG AAG CAT AAA TGTTTC AGG AAG GAA CTC GAA 1168 Leu Phe Met Gly Asn Leu Lys His Lys Cys PheArg Lys Glu Leu Glu 1280 1285 1290 GAG AAT GAA ACA TTA GAA AGT ATC ATGAAT ACT GCT GAG AGT GAA GAA 1216 Glu Asn Glu Thr Leu Glu Ser Ile Met AsnThr Ala Glu Ser Glu Glu 1295 1300 1305 GAA TTG AAA AAA TAT TTT TAT TACTTG GAG GGA TCC AAA GAT GCT CTA 1264 Glu Leu Lys Lys Tyr Phe Tyr Tyr LeuGlu Gly Ser Lys Asp Ala Leu 1310 1315 1320 CTC TGC GGC TTC AGC ACA GATTCA GGG CAG TGT CCA GAA GGC TAC ATC 1312 Leu Cys Gly Phe Ser Thr Asp SerGly Gln Cys Pro Glu Gly Tyr Ile 1325 1330 1335 1340 TGT GTG AAG GCT GGCAGA AAC CCG GAT TAT GGC TAC ACG AGC TTT GAC 1360 Cys Val Lys Ala Gly ArgAsn Pro Asp Tyr Gly Tyr Thr Ser Phe Asp 1345 1350 1355 ACA TTC AGC TGGGCC TTC TTG GCC TTG TTT CGG CTA ATG ACT CAG GAC 1408 Thr Phe Ser Trp AlaPhe Leu Ala Leu Phe Arg Leu Met Thr Gln Asp 1360 1365 1370 TAC TGG GAGAAC CTT TAC CAA CAG ACT CTG CGT GCT GCT GGC AAA ACC 1456 Tyr Trp Glu AsnLeu Tyr Gln Gln Thr Leu Arg Ala Ala Gly Lys Thr 1375 1380 1385 TAC ATGATT TTC TTT GTC GTG GTT ATT TTT CTG GGC TCC TTT TAC CTG 1504 Tyr Met IlePhe Phe Val Val Val Ile Phe Leu Gly Ser Phe Tyr Leu 1390 1395 1400 ATAAAC TTG ATC CTG GCT GTG GTA GCC ATG GCG TAT GAG GAA CAG AAC 1552 Ile AsnLeu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu Gln Asn 1405 1410 14151420 CAG GCC AAC ATC GAA GAA GCT AAA CAG AAA GAG TTA GAA TTT CAG CAG1600 Gln Ala Asn Ile Glu Glu Ala Lys Gln Lys Glu Leu Glu Phe Gln Gln1425 1430 1435 ATG TTA GAC CGA CTC AAA AAG GAG CAG GAA GAA GCT GAG GCGATC GCT 1648 Met Leu Asp Arg Leu Lys Lys Glu Gln Glu Glu Ala Glu Ala IleAla 1440 1445 1450 GCA GCT GCT GCT GAG TTC ACG AGT ATA GGG CGG AGC AGGATC ATG GGA 1696 Ala Ala Ala Ala Glu Phe Thr Ser Ile Gly Arg Ser Arg IleMet Gly 1455 1460 1465 CTC TCT GAG AGC TCT TCA GAA ACC TCC AGG CTG AGCTCA AAG AGT GCC 1744 Leu Ser Glu Ser Ser Ser Glu Thr Ser Arg Leu Ser SerLys Ser Ala 1470 1475 1480 AAG GAG AGA AGA AAC CGA AGA AAG AAA AAG AAACAG AAG ATG TCC AGT 1792 Lys Glu Arg Arg Asn Arg Arg Lys Lys Lys Lys GlnLys Met Ser Ser 1485 1490 1495 1500 GGC GAG GAA AAG GGT GAC GAT GAG AAGCTG TCC AAG TCA GGA TCA GAG 1840 Gly Glu Glu Lys Gly Asp Asp Glu Lys LeuSer Lys Ser Gly Ser Glu 1505 1510 1515 GAA AGC ATC CGA AAG AAA AGC TTCCAT CTC GGT GTG GAA GGG CAC CAC 1888 Glu Ser Ile Arg Lys Lys Ser Phe HisLeu Gly Val Glu Gly His His 1520 1525 1530 CGG ACC CGG GAA AAG AGG CTGTCC ACC CCC AAC CAG TCG CCA CTC AGC 1936 Arg Thr Arg Glu Lys Arg Leu SerThr Pro Asn Gln Ser Pro Leu Ser 1535 1540 1545 ATT CGC GGG TCC CTG TTTTCT GCC AGG CGC AGC AGC AGG ACG AGT CTC 1984 Ile Arg Gly Ser Leu Phe SerAla Arg Arg Ser Ser Arg Thr Ser Leu 1550 1555 1560 TTC AGT TTT AAG GGGCGA GGA AGA GAT CTG GGA TCT GAG ACA GAA TTC 2032 Phe Ser Phe Lys Gly ArgGly Arg Asp Leu Gly Ser Glu Thr Glu Phe 1565 1570 1575 1580 GCT GAT GATGAG CAT AGC ATT TTT GGA GAC AAC GAG AGC AGA AGG GGT 2080 Ala Asp Asp GluHis Ser Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly 1585 1590 1595 TCA CTATTC GTA CCC CAT AGA CCC CGG GAG CGG CGC AGC AGT AAC ATC 2128 Ser Leu PheVal Pro His Arg Pro Arg Glu Arg Arg Ser Ser Asn Ile 1600 1605 1610 AGTCAG GCC AGT AGG TCC CCG CCA GTG CTA CCG GTG AAC GGG AAG ATG 2176 Ser GlnAla Ser Arg Ser Pro Pro Val Leu Pro Val Asn Gly Lys Met 1615 1620 1625CAC AGT GCA GTG GAC TGC AAT GGA GTC GTG TCG CTT GTT GAT GGA CCC 2224 HisSer Ala Val Asp Cys Asn Gly Val Val Ser Leu Val Asp Gly Pro 1630 16351640 TCA GCC CTC ATG CTC CCC AAT GGA CAG CTT CTT CCA GAG GTG ATA ATA2272 Ser Ala Leu Met Leu Pro Asn Gly Gln Leu Leu Pro Glu Val Ile Ile1645 1650 1655 1660 GAT AAG GCA ACT TCC GAC GAC AGC GGC ACG ACT AAT CAGATG CGC AAA 2320 Asp Lys Ala Thr Ser Asp Asp Ser Gly Thr Thr Asn Gln MetArg Lys 1665 1670 1675 AAA AGG CTC TCT AGT TCT TAC TTC TTG TCT GAG GACATG CTG AAT GAC 2368 Lys Arg Leu Ser Ser Ser Tyr Phe Leu Ser Glu Asp MetLeu Asn Asp 1680 1685 1690 CCG CAT CTC AGG CAA AGG GCC ATG AGC AGG GCGAGC ATA CTG ACC AAC 2416 Pro His Leu Arg Gln Arg Ala Met Ser Arg Ala SerIle Leu Thr Asn 1695 1700 1705 ACT GTG GAA GAA CTT GAA GAA TCT AGA CAAAAA TGT CCA CCA TGG TGG 2464 Thr Val Glu Glu Leu Glu Glu Ser Arg Gln LysCys Pro Pro Trp Trp 1710 1715 1720 TAC AGA TTT GCT CAC ACA TTT TTA ATCTGG AAT TGC TCT CCA TAT TGG 2512 Tyr Arg Phe Ala His Thr Phe Leu Ile TrpAsn Cys Ser Pro Tyr Trp 1725 1730 1735 1740 ATA AAA TTC AAA AAG CTC ATCTAT TTT ATT GTG ATG GAT CCT TTT GTA 2560 Ile Lys Phe Lys Lys Leu Ile TyrPhe Ile Val Met Asp Pro Phe Val 1745 1750 1755 GAT CTT GCA ATT ACC ATTTGC ATA GTT TTA AAC ACC TTA TTT ATG GCT 2608 Asp Leu Ala Ile Thr Ile CysIle Val Leu Asn Thr Leu Phe Met Ala 1760 1765 1770 ATG GAG CAC CAC CCAATG ACT GAA GAA TTC AAA AAT GTC CTT GCA GTG 2656 Met Glu His His Pro MetThr Glu Glu Phe Lys Asn Val Leu Ala Val 1775 1780 1785 GGG AAC TTG ATCTTT ACA GGG ATC TTC GCA GCT GAA ATG GTA CTG AAG 2704 Gly Asn Leu Ile PheThr Gly Ile Phe Ala Ala Glu Met Val Leu Lys 1790 1795 1800 TTA ATA GCCATG GAC CCC TAT GAG TAT TTC CAA GTA GGG TGG AAT ATT 2752 Leu Ile Ala MetAsp Pro Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile 1805 1810 1815 1820 TTTGAC AGC CTA ATT GTG ACG CTG AGT TTG ATA GAG CTT TTC CTA GCA 2800 Phe AspSer Leu Ile Val Thr Leu Ser Leu Ile Glu Leu Phe Leu Ala 1825 1830 1835GAT GTG GAA GGA TTA TCA GTT CTG CGG TCA TTC AGA TTG CTC CGA GTC 2848 AspVal Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val 1840 18451850 TTC AAG TTG GCA AAG TCC TGG CCC ACA CTG AAC ATG CTC ATT AAG ATC2896 Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile1855 1860 1865 ATC GGC AAC TCG GTG GGC GCA CTG GGC AAC CTG ACC CTG GTGCTG GCC 2944 Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val LeuAla 1870 1875 1880 ATC ATC GTC TTC ATT TTT GCC GTG GTC GGC ATG CAG CTGTTT GGA AAG 2992 Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu PheGly Lys 1885 1890 1895 1900 AGC TAC AAG GAG TGT GTC TGC AAG ATC AAT GTGGAC TGC AAG CTG CCG 3040 Ser Tyr Lys Glu Cys Val Cys Lys Ile Asn Val AspCys Lys Leu Pro 1905 1910 1915 CGC TGG CAC ATG AAC GAC TTC TTC CAC TCCTTC CTC ATC GTG TTC CGA 3088 Arg Trp His Met Asn Asp Phe Phe His Ser PheLeu Ile Val Phe Arg 1920 1925 1930 GTG CTG TGT GGG GAG TGG ATA GAG ACCATG TGG GAC TGC ATG GAG GTC 3136 Val Leu Cys Gly Glu Trp Ile Glu Thr MetTrp Asp Cys Met Glu Val 1935 1940 1945 GCG GGC CAG ACC ATG TGC CTT ATTGTT TAC ATG ATG GTC ATG GTG ATT 3184 Ala Gly Gln Thr Met Cys Leu Ile ValTyr Met Met Val Met Val Ile 1950 1955 1960 GGG AAC CTT GTG GTC CTG AACCTG TTT CTG GCT CTT TTG CTG AGT TCC 3232 Gly Asn Leu Val Val Leu Asn LeuPhe Leu Ala Leu Leu Leu Ser Ser 1965 1970 1975 1980 TTT AGT TCT GAC AATCTT ACA GCA ATT GAG GAA GAC ACC GAT GCA AAC 3280 Phe Ser Ser Asp Asn LeuThr Ala Ile Glu Glu Asp Thr Asp Ala Asn 1985 1990 1995 AAC CTC CAG ATCGCA GTG GCC AGA ATT AAG AGG GGA ATC AAT TAC GTG 3328 Asn Leu Gln Ile AlaVal Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val 2000 2005 2010 AAA CAG ACCCTG CGT GAA TTC ATT CTA AAA TCA TTT TCC AAA AAG CCA 3376 Lys Gln Thr LeuArg Glu Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro 2015 2020 2025 AAG GGCTCC AAG GAC ACA AAA CGA ACA GCA GAT CCC AAC AAC AAG AAA 3424 Lys Gly SerLys Asp Thr Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys 2030 2035 2040 GAAAAC TAT ATT TCA AAC CGT ACC CTT GCG GAG ATG AGC AAG GAT CAC 3472 Glu AsnTyr Ile Ser Asn Arg Thr Leu Ala Glu Met Ser Lys Asp His 2045 2050 20552060 AAT TTC CTC AAA GAA AAG GAT AGG ATC AGT GGT TAT GGC AGC AGT CTA3520 Asn Phe Leu Lys Glu Lys Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu2065 2070 2075 GAC AAA AGC TTT ATG GAT GAA AAT GAT TAC CAG TCC TTT ATCCAT AAC 3568 Asp Lys Ser Phe Met Asp Glu Asn Asp Tyr Gln Ser Phe Ile HisAsn 2080 2085 2090 CCC AGC CTC ACA GTG ACA GTG CCA ATT GCA CCT GGG GAGTCT GAT TTG 3616 Pro Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu SerAsp Leu 2095 2100 2105 GAG ATT ATG AAC ACA GAA GAG CTT AGC AGT GAC TCAGAC AGT GAC TAC 3664 Glu Ile Met Asn Thr Glu Glu Leu Ser Ser Asp Ser AspSer Asp Tyr 2110 2115 2120 AGC AAA GAG AAA CGG AAC CGA TCA AGC TCT TCTGAG TGC AGC ACT GTT 3712 Ser Lys Glu Lys Arg Asn Arg Ser Ser Ser Ser GluCys Ser Thr Val 2125 2130 2135 2140 GAC AAC CCT CTG CCA GGA GAA GAG GAGGCT GAA GCA GAG CCC GTA AAC 3760 Asp Asn Pro Leu Pro Gly Glu Glu Glu AlaGlu Ala Glu Pro Val Asn 2145 2150 2155 GCA GAT GAG CCT GAA GCC TGC TTTACA GAT GGT TGT GTG AGG AGA TTT 3808 Ala Asp Glu Pro Glu Ala Cys Phe ThrAsp Gly Cys Val Arg Arg Phe 2160 2165 2170 CCA TGC TGC CAA GTT AAT GTAGAC TCT GGG AAA GGG AAA GTT TGG TGG 3856 Pro Cys Cys Gln Val Asn Val AspSer Gly Lys Gly Lys Val Trp Trp 2175 2180 2185 ACC ATC AGG AAG ACG TGCTAC AGG ATA GTT GAA CAC AGC TGG TTT GAA 3904 Thr Ile Arg Lys Thr Cys TyrArg Ile Val Glu His Ser Trp Phe Glu 2190 2195 2200 AGC TTC ATC GTT CTCATG ATC CTG CTC AGC AGT GGA GCT CTG GCT TTT 3952 Ser Phe Ile Val Leu MetIle Leu Leu Ser Ser Gly Ala Leu Ala Phe 2205 2210 2215 2220 GAA GAT ATCTAT ATT GAA AAG AAA AAG ACC ATT AAG ATT ATC CTG GAG 4000 Glu Asp Ile TyrIle Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu 2225 2230 2235 TAT GCTGAC AAG ATA TTC ACC TAC ATC TTC ATT CTG GAA ATG CTT CTA 4048 Tyr Ala AspLys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 2240 2245 2250 AAATGG GTC GCA TAT GGG TAT AAA ACA TAT TTC ACT AAT GCC TGG TGT 4096 Lys TrpVal Ala Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys 2255 2260 2265TGG CTG GAC TTC TTA ATT GTT GAT GTG TCT CTA GTT ACT TTA GTA GCC 4144 TrpLeu Asp Phe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala 2270 22752280 AAC ACT CTT GGC TAC TCA GAC CTT GGC CCC ATT AAA TCT CTA CGG ACA4192 Asn Thr Leu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr2285 2290 2295 2300 CTG AGG GCC CTA AGA CCC CTA AGA GCC TTG TCT AGA TTTGAA GGA ATG 4240 Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe GluGly Met 2305 2310 2315 AGG GTA GTG GTC AAC GCA CTC ATA GGA GCA ATC CCTTCC ATC ATG AAC 4288 Arg Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro SerIle Met Asn 2320 2325 2330 GTG CTT CTC GTG TGC CTT ATA TTC TGG CTA ATATTT AGC ATC ATG GGA 4336 Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile PheSer Ile Met Gly 2335 2340 2345 GTC AAT CTG TTT GCT GGC AAG TTC TAT GAGTGT GTC AAC ACC ACC GAT 4384 Val Asn Leu Phe Ala Gly Lys Phe Tyr Glu CysVal Asn Thr Thr Asp 2350 2355 2360 GGG TCA CGA TTT CCT ACA TCT CAA GTTGCA AAC CGT TCT GAG TGT TTT 4432 Gly Ser Arg Phe Pro Thr Ser Gln Val AlaAsn Arg Ser Glu Cys Phe 2365 2370 2375 2380 GCC CTG ATG AAC GTT AGT GGAAAT GTG CGA TGG AAA AAC CTG AAA GTA 4480 Ala Leu Met Asn Val Ser Gly AsnVal Arg Trp Lys Asn Leu Lys Val 2385 2390 2395 AAC TTC GAC AAC GTT GGGCTT GGT TAC CTG TCG CTG CTT CAA GTT GCA 4528 Asn Phe Asp Asn Val Gly LeuGly Tyr Leu Ser Leu Leu Gln Val Ala 2400 2405 2410 ACA TTC AAG GGC TGGATG GAT ATT ATG TAT GCA GCA GTT GAC TCT GTT 4576 Thr Phe Lys Gly Trp MetAsp Ile Met Tyr Ala Ala Val Asp Ser Val 2415 2420 2425 AAT GTA AAT GAACAG CCG AAA TAC GAA TAC AGT CTC TAC ATG TAC ATT 4624 Asn Val Asn Glu GlnPro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile 2430 2435 2440 TAC TTT GTCATC TTC ATC ATC TTC GGC TCA TTC TTC ACG TTG AAC CTG 4672 Tyr Phe Val IlePhe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 2445 2450 2455 2460 TTCATT GGT GTC ATC ATA GAT AAT TTC AAC CAA CAG AAA AAA AAG CTT 4720 Phe IleGly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu 2465 2470 2475GGA GGT CAA GAT ATC TTT ATG ACA GAA GAA CAG AAG AAA TAC TAT AAT 4768 GlyGly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 2480 24852490 GCA ATG AAG AAG CTT GGG TCC AAA AAA CCA CAA AAA CCA ATT CCA AGG4816 Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg2495 2500 2505 CCA GGG AAC AAA TTC CAA GGA TGT ATA TTT GAC TTA GTG ACAAAC CAA 4864 Pro Gly Asn Lys Phe Gln Gly Cys Ile Phe Asp Leu Val Thr AsnGln 2510 2515 2520 GCT TTT GAT ATC ACC ATC ATG GTT CTT ATA TGC CTC AACATG GTA ACC 4912 Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu Asn MetVal Thr 2525 2530 2535 2540 ATG ATG GTA GAA AAA GAG GGG CAA ACT GAG TACATG GAT TAT GTT TTA 4960 Met Met Val Glu Lys Glu Gly Gln Thr Glu Tyr MetAsp Tyr Val Leu 2545 2550 2555 CAC TGG ATC AAC ATG GTC TTC ATT ATC CTGTTC ACT GGG GAG TGT GTG 5008 His Trp Ile Asn Met Val Phe Ile Ile Leu PheThr Gly Glu Cys Val 2560 2565 2570 CTG AAG CTA ATC TCC CTC AGA CAT TACTAC TTC ACT GTG GGT TGG AAC 5056 Leu Lys Leu Ile Ser Leu Arg His Tyr TyrPhe Thr Val Gly Trp Asn 2575 2580 2585 ATT TTT GAT TTT GTG GTA GTG ATCCTC TCC ATT GTA GGA ATG TTT CTC 5104 Ile Phe Asp Phe Val Val Val Ile LeuSer Ile Val Gly Met Phe Leu 2590 2595 2600 GCT GAG ATG ATA GAG AAG TATTTC GTG TCC CCT ACC CTG TTC CGA GTC 5152 Ala Glu Met Ile Glu Lys Tyr PheVal Ser Pro Thr Leu Phe Arg Val 2605 2610 2615 2620 ATC CGC CTG GCC AGGATT GGA CGA ATC CTA CGC CTG ATC AAA GGC GCC 5200 Ile Arg Leu Ala Arg IleGly Arg Ile Leu Arg Leu Ile Lys Gly Ala 2625 2630 2635 AAG GGG ATC CGCACT CTG CTC TTT GCT TTG ATG ATG TCC CTT CCT GCG 5248 Lys Gly Ile Arg ThrLeu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 2640 2645 2650 CTG TTC AACATC GGC CTC CTG CTT TTC CTG GTC ATG TTC ATC TAC GCC 5296 Leu Phe Asn IleGly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala 2655 2660 2665 ATC TTTGGG ATG TCC AAC TTT GCC TAC GTT AAA AAG GAG GCT GGA ATT 5344 Ile Phe GlyMet Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile 2670 2675 2680 AATGAC ATG TTC AAC TTT GAG ACT TTT GGC AAC AGC ATG ATC TGC TTG 5392 Asn AspMet Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu 2685 2690 26952700 TTC CAA ATC ACC ACC TCT GCC GGC TGG GAC GGA CTG CTG GCC CCC ATC5440 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile2705 2710 2715 CTC AAC AGC GCA CCT CCC GAC TGT GAC CCT AAA AAA GTT CACCCA GGA 5488 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro Lys Lys Val His ProGly 2720 2725 2730 AGT TCA GTG GAA GGG GAC TGT GGG AAC CCA TCC GTG GGGATT TTT TAC 5536 Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly IlePhe Tyr 2735 2740 2745 TTT GTC AGC TAC ATC ATC ATA TCC TTC CTG GTG GTGGTG AAC ATG TAC 5584 Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val ValAsn Met Tyr 2750 2755 2760 ATC GCT GTC ATC CTG GAG AAC TTC AGC GTC GCCACC GAA GAG AGC ACT 5632 Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala ThrGlu Glu Ser Thr 2765 2770 2775 2780 GAG CCT CTG AGT GAG GAC GAC TTT GAGATG TTC TAC GAG GTC TGG GAG 5680 Glu Pro Leu Ser Glu Asp Asp Phe Glu MetPhe Tyr Glu Val Trp Glu 2785 2790 2795 AAG TTC GAC CCT GAC GCC ACT CAGTTC ATA GAG TTC TGC AAG CTC TCT 5728 Lys Phe Asp Pro Asp Ala Thr Gln PheIle Glu Phe Cys Lys Leu Ser 2800 2805 2810 GAC TTT GCA GCT GCC CTG GATCCT CCC CTC CTC ATC GCA AAG CCA AAC 5776 Asp Phe Ala Ala Ala Leu Asp ProPro Leu Leu Ile Ala Lys Pro Asn 2815 2820 2825 AAA GTC CAG CTC ATT GCCATG GAC CTG CCC ATG GTG AGT GGA GAC CGC 5824 Lys Val Gln Leu Ile Ala MetAsp Leu Pro Met Val Ser Gly Asp Arg 2830 2835 2840 ATC CAC TGC CTG GACATC TTG TTT GCT TTT ACA AAG CGG GTC CTG GGT 5872 Ile His Cys Leu Asp IleLeu Phe Ala Phe Thr Lys Arg Val Leu Gly 2845 2850 2855 2860 GAG GGT GGAGAG ATG GAT TCT CTT CGT TCA CAG ATG GAA GAA AGG TTC 5920 Glu Gly Gly GluMet Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe 2865 2870 2875 ATG TCAGCC AAT CCT TCT AAA GTG TCC TAT GAA CCC ATC ACG ACC ACA 5968 Met Ser AlaAsn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr 2880 2885 2890 CTGAAG AGA AAA CAA GAG GAG GTG TCC GCG ACT ATC ATT CAG CGT GCT 6016 Leu LysArg Lys Gln Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala 2895 2900 2905TAC AGA CGG TAT CGC CTC AGA CAA CAC GTC AAG AAT ATA TCG AGT ATA 6064 TyrArg Arg Tyr Arg Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile 2910 29152920 TAC ATA AAA GAT GGA GAC AGG GAT GAT GAT TTG CCC AAT AAA GAA GAT6112 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp2925 2930 2935 2940 ACA GTT TTT GAT AAC GTG AAC GAG AAC TCA AGT CCG GAAAAG ACA GAT 6160 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser Pro Glu LysThr Asp 2945 2950 2955 GTA ACT GCC TCA ACC ATC TCG CCA CCT TCC TAT GACAGT GTC ACA AAG 6208 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser Tyr Asp SerVal Thr Lys 2960 2965 2970 CCA GAT CAA GAG AAA TAT GAA ACA GAC AAA ACAGAG AAG GAA GAC AAA 6256 Pro Asp Gln Glu Lys Tyr Glu Thr Asp Lys Thr GluLys Glu Asp Lys 2975 2980 2985 GAG AAA GAT GAA AGC AGG AAA TAGAGCTTTGGTTTTGATAC ACTGTTGACA 6307 Glu Lys Asp Glu Ser Arg Lys 2990 2995GCCTGTGAAG GTTGACTCAC TCGTGTTAGT AAGACTCTTT TACGGAGGTC TATCCAAACT 6367CTTTTATCAA AAATTCTCAA GGCAGCACAG CCATTAGCTC TGATCCAACG AGGCAGAGGG 6427CAGCATTTAC ACATGGCTAT GTTTT 6452 1984 amino acids amino acid linearprotein 10 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His PheThr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Ser Glu GluLys Ala 20 25 30 Lys Glu His Lys Asp Glu Lys Lys Asp Asp Glu Glu Glu GlyPro Lys 35 40 45 Pro Ser Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe IleTyr Gly 50 55 60 Asp Ile Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp LeuAsp Pro 65 70 75 80 Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn LysGly Lys Ala 85 90 95 Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met LeuSer Pro Phe 100 105 110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu ValHis Ser Leu Phe 115 120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr AsnCys Ile Phe Met Thr 130 135 140 Leu Ser Asn Pro Pro Glu Trp Thr Lys AsnVal Glu Tyr Thr Phe Thr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser LeuIle Lys Ile Leu Ala Arg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr PheLeu Arg Asp Pro Trp Asn Trp Leu Asp 180 185 190 Phe Val Val Ile Val PheAla Tyr Leu Thr Glu Phe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala LeuArg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val IlePro Gly Leu Lys Thr Ile Val Gly Ala Leu Ile Gln 225 230 235 240 Ser ValLys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe Cys Leu 245 250 255 SerVal Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270His Lys Cys Phe Arg Lys Glu Leu Glu Glu Asn Glu Thr Leu Glu Ser 275 280285 Ile Met Asn Thr Ala Glu Ser Glu Glu Glu Leu Lys Lys Tyr Phe Tyr 290295 300 Tyr Leu Glu Gly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp305 310 315 320 Ser Gly Gln Cys Pro Glu Gly Tyr Ile Cys Val Lys Ala GlyArg Asn 325 330 335 Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser TrpAla Phe Leu 340 345 350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp GluAsn Leu Tyr Gln 355 360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr MetIle Phe Phe Val Val 370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu IleAsn Leu Ile Leu Ala Val 385 390 395 400 Val Ala Met Ala Tyr Glu Glu GlnAsn Gln Ala Asn Ile Glu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu PheGln Gln Met Leu Asp Arg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala GluAla Ile Ala Ala Ala Ala Ala Glu Phe Thr 435 440 445 Ser Ile Gly Arg SerArg Ile Met Gly Leu Ser Glu Ser Ser Ser Glu 450 455 460 Thr Ser Arg LeuSer Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys LysLys Lys Gln Lys Met Ser Ser Gly Glu Glu Lys Gly Asp Asp 485 490 495 GluLys Leu Ser Lys Ser Gly Ser Glu Glu Ser Ile Arg Lys Lys Ser 500 505 510Phe His Leu Gly Val Glu Gly His His Arg Thr Arg Glu Lys Arg Leu 515 520525 Ser Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe Ser 530535 540 Ala Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg Gly545 550 555 560 Arg Asp Leu Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu HisSer Ile 565 570 575 Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe ValPro His Arg 580 585 590 Pro Arg Glu Arg Arg Ser Ser Asn Ile Ser Gln AlaSer Arg Ser Pro 595 600 605 Pro Val Leu Pro Val Asn Gly Lys Met His SerAla Val Asp Cys Asn 610 615 620 Gly Val Val Ser Leu Val Asp Gly Pro SerAla Leu Met Leu Pro Asn 625 630 635 640 Gly Gln Leu Leu Pro Glu Val IleIle Asp Lys Ala Thr Ser Asp Asp 645 650 655 Ser Gly Thr Thr Asn Gln MetArg Lys Lys Arg Leu Ser Ser Ser Tyr 660 665 670 Phe Leu Ser Glu Asp MetLeu Asn Asp Pro His Leu Arg Gln Arg Ala 675 680 685 Met Ser Arg Ala SerIle Leu Thr Asn Thr Val Glu Glu Leu Glu Glu 690 695 700 Ser Arg Gln LysCys Pro Pro Trp Trp Tyr Arg Phe Ala His Thr Phe 705 710 715 720 Leu IleTrp Asn Cys Ser Pro Tyr Trp Ile Lys Phe Lys Lys Leu Ile 725 730 735 TyrPhe Ile Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr Ile Cys 740 745 750Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His His Pro Met Thr 755 760765 Glu Glu Phe Lys Asn Val Leu Ala Val Gly Asn Leu Ile Phe Thr Gly 770775 780 Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro Tyr785 790 795 800 Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu IleVal Thr 805 810 815 Leu Ser Leu Ile Glu Leu Phe Leu Ala Asp Val Glu GlyLeu Ser Val 820 825 830 Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys LeuAla Lys Ser Trp 835 840 845 Pro Thr Leu Asn Met Leu Ile Lys Ile Ile GlyAsn Ser Val Gly Ala 850 855 860 Leu Gly Asn Leu Thr Leu Val Leu Ala IleIle Val Phe Ile Phe Ala 865 870 875 880 Val Val Gly Met Gln Leu Phe GlyLys Ser Tyr Lys Glu Cys Val Cys 885 890 895 Lys Ile Asn Val Asp Cys LysLeu Pro Arg Trp His Met Asn Asp Phe 900 905 910 Phe His Ser Phe Leu IleVal Phe Arg Val Leu Cys Gly Glu Trp Ile 915 920 925 Glu Thr Met Trp AspCys Met Glu Val Ala Gly Gln Thr Met Cys Leu 930 935 940 Ile Val Tyr MetMet Val Met Val Ile Gly Asn Leu Val Val Leu Asn 945 950 955 960 Leu PheLeu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu Thr 965 970 975 AlaIle Glu Glu Asp Thr Asp Ala Asn Asn Leu Gln Ile Ala Val Ala 980 985 990Arg Ile Lys Arg Gly Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu Phe 995 10001005 Ile Leu Lys Ser Phe Ser Lys Lys Pro Lys Gly Ser Lys Asp Thr Lys1010 1015 1020 Arg Thr Ala Asp Pro Asn Asn Lys Lys Glu Asn Tyr Ile SerAsn Arg 1025 1030 1035 1040 Thr Leu Ala Glu Met Ser Lys Asp His Asn PheLeu Lys Glu Lys Asp 1045 1050 1055 Arg Ile Ser Gly Tyr Gly Ser Ser LeuAsp Lys Ser Phe Met Asp Glu 1060 1065 1070 Asn Asp Tyr Gln Ser Phe IleHis Asn Pro Ser Leu Thr Val Thr Val 1075 1080 1085 Pro Ile Ala Pro GlyGlu Ser Asp Leu Glu Ile Met Asn Thr Glu Glu 1090 1095 1100 Leu Ser SerAsp Ser Asp Ser Asp Tyr Ser Lys Glu Lys Arg Asn Arg 1105 1110 1115 1120Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly Glu 11251130 1135 Glu Glu Ala Glu Ala Glu Pro Val Asn Ala Asp Glu Pro Glu AlaCys 1140 1145 1150 Phe Thr Asp Gly Cys Val Arg Arg Phe Pro Cys Cys GlnVal Asn Val 1155 1160 1165 Asp Ser Gly Lys Gly Lys Val Trp Trp Thr IleArg Lys Thr Cys Tyr 1170 1175 1180 Arg Ile Val Glu His Ser Trp Phe GluSer Phe Ile Val Leu Met Ile 1185 1190 1195 1200 Leu Leu Ser Ser Gly AlaLeu Ala Phe Glu Asp Ile Tyr Ile Glu Lys 1205 1210 1215 Lys Lys Thr IleLys Ile Ile Leu Glu Tyr Ala Asp Lys Ile Phe Thr 1220 1225 1230 Tyr IlePhe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Tyr 1235 1240 1245Lys Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val 12501255 1260 Asp Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr SerAsp 1265 1270 1275 1280 Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg AlaLeu Arg Pro Leu 1285 1290 1295 Arg Ala Leu Ser Arg Phe Glu Gly Met ArgVal Val Val Asn Ala Leu 1300 1305 1310 Ile Gly Ala Ile Pro Ser Ile MetAsn Val Leu Leu Val Cys Leu Ile 1315 1320 1325 Phe Trp Leu Ile Phe SerIle Met Gly Val Asn Leu Phe Ala Gly Lys 1330 1335 1340 Phe Tyr Glu CysVal Asn Thr Thr Asp Gly Ser Arg Phe Pro Thr Ser 1345 1350 1355 1360 GlnVal Ala Asn Arg Ser Glu Cys Phe Ala Leu Met Asn Val Ser Gly 1365 13701375 Asn Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val Gly Leu1380 1385 1390 Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly TrpMet Asp 1395 1400 1405 Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val AsnGlu Gln Pro Lys 1410 1415 1420 Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile TyrPhe Val Ile Phe Ile Ile 1425 1430 1435 1440 Phe Gly Ser Phe Phe Thr LeuAsn Leu Phe Ile Gly Val Ile Ile Asp 1445 1450 1455 Asn Phe Asn Gln GlnLys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met 1460 1465 1470 Thr Glu GluGln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser 1475 1480 1485 LysLys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly 1490 14951500 Cys Ile Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Thr Ile Met1505 1510 1515 1520 Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val GluLys Glu Gly 1525 1530 1535 Gln Thr Glu Tyr Met Asp Tyr Val Leu His TrpIle Asn Met Val Phe 1540 1545 1550 Ile Ile Leu Phe Thr Gly Glu Cys ValLeu Lys Leu Ile Ser Leu Arg 1555 1560 1565 His Tyr Tyr Phe Thr Val GlyTrp Asn Ile Phe Asp Phe Val Val Val 1570 1575 1580 Ile Leu Ser Ile ValGly Met Phe Leu Ala Glu Met Ile Glu Lys Tyr 1585 1590 1595 1600 Phe ValSer Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly 1605 1610 1615Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu 16201625 1630 Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly LeuLeu 1635 1640 1645 Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly MetSer Asn Phe 1650 1655 1660 Ala Tyr Val Lys Lys Glu Ala Gly Ile Asn AspMet Phe Asn Phe Glu 1665 1670 1675 1680 Thr Phe Gly Asn Ser Met Ile CysLeu Phe Gln Ile Thr Thr Ser Ala 1685 1690 1695 Gly Trp Asp Gly Leu LeuAla Pro Ile Leu Asn Ser Ala Pro Pro Asp 1700 1705 1710 Cys Asp Pro LysLys Val His Pro Gly Ser Ser Val Glu Gly Asp Cys 1715 1720 1725 Gly AsnPro Ser Val Gly Ile Phe Tyr Phe Val Ser Tyr Ile Ile Ile 1730 1735 1740Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 17451750 1755 1760 Phe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser GluAsp Asp 1765 1770 1775 Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe AspPro Asp Ala Thr 1780 1785 1790 Gln Phe Ile Glu Phe Cys Lys Leu Ser AspPhe Ala Ala Ala Leu Asp 1795 1800 1805 Pro Pro Leu Leu Ile Ala Lys ProAsn Lys Val Gln Leu Ile Ala Met 1810 1815 1820 Asp Leu Pro Met Val SerGly Asp Arg Ile His Cys Leu Asp Ile Leu 1825 1830 1835 1840 Phe Ala PheThr Lys Arg Val Leu Gly Glu Gly Gly Glu Met Asp Ser 1845 1850 1855 LeuArg Ser Gln Met Glu Glu Arg Phe Met Ser Ala Asn Pro Ser Lys 1860 18651870 Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln Glu Glu1875 1880 1885 Val Ser Ala Thr Ile Ile Gln Arg Ala Tyr Arg Arg Tyr ArgLeu Arg 1890 1895 1900 Gln His Val Lys Asn Ile Ser Ser Ile Tyr Ile LysAsp Gly Asp Arg 1905 1910 1915 1920 Asp Asp Asp Leu Pro Asn Lys Glu AspThr Val Phe Asp Asn Val Asn 1925 1930 1935 Glu Asn Ser Ser Pro Glu LysThr Asp Val Thr Ala Ser Thr Ile Ser 1940 1945 1950 Pro Pro Ser Tyr AspSer Val Thr Lys Pro Asp Gln Glu Lys Tyr Glu 1955 1960 1965 Thr Asp LysThr Glu Lys Glu Asp Lys Glu Lys Asp Glu Ser Arg Lys 1970 1975 1980 1989amino acids amino acid Not Relevant Not Relevant protein 11 Met Ala MetLeu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys GlnSer Leu Ala Leu Ile Glu Gln Arg Ile Xaa Glu Xaa Lys Xaa 20 25 30 Lys GluXaa Lys Xaa Glu Lys Lys Asp Asp Xaa Glu Glu Xaa Pro Lys 35 40 45 Pro SerSer Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp IlePro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 TyrTyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Xaa 85 90 95 IlePhe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130135 140 Xaa Xaa Asn Pro Pro Xaa Trp Thr Lys Asn Val Xaa Tyr Thr Phe Thr145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Xaa Lys Ile Leu Ala ArgGly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp AsnTrp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu PheVal Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val LeuArg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr IleVal Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp ValMet Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile GlyLeu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg XaaXaa Leu Glu Xaa Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr XaaGlu Ser Glu Glu Xaa Xaa Xaa Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu GlySer Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser GlyGln Cys Pro Glu Gly Tyr Xaa Cys Val Lys Xaa Gly Arg Asn 325 330 335 ProAsp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile GluGlu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp ArgLeu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala AlaAla Glu Xaa Thr 435 440 445 Ser Ile Xaa Arg Ser Arg Ile Met Gly Leu SerGlu Ser Ser Ser Glu 450 455 460 Thr Ser Xaa Leu Ser Ser Lys Ser Ala LysGlu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Xaa Gln Lys Lys XaaSer Ser Gly Glu Glu Lys Gly Asp 485 490 495 Xaa Glu Lys Leu Ser Lys SerXaa Ser Glu Xaa Ser Ile Arg Xaa Lys 500 505 510 Ser Phe His Leu Gly ValGlu Gly His Xaa Arg Xaa Xaa Glu Lys Arg 515 520 525 Leu Ser Thr Pro AsnGln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala Arg ArgSer Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 Gly ArgAsp Xaa Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575 IlePhe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585 590Arg Pro Xaa Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595 600605 Pro Pro Xaa Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys 610615 620 Asn Gly Val Val Ser Leu Val Asp Gly Xaa Ser Ala Leu Met Leu Pro625 630 635 640 Asn Gly Gln Leu Leu Pro Glu Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 645 650 655 Xaa Xaa Gly Thr Thr Asn Gln Xaa Xaa Lys Lys Arg XaaXaa Ser Ser 660 665 670 Tyr Xaa Leu Ser Glu Asp Met Leu Asn Asp Pro XaaLeu Arg Gln Arg 675 680 685 Ala Met Ser Arg Ala Ser Ile Leu Thr Asn ThrVal Glu Glu Leu Glu 690 695 700 Glu Ser Arg Gln Lys Cys Xaa Xaa Xaa XaaTyr Arg Phe Ala His Xaa 705 710 715 720 Phe Leu Ile Trp Asn Cys Ser ProTyr Trp Ile Lys Phe Lys Lys Xaa 725 730 735 Ile Tyr Phe Ile Val Met AspPro Phe Val Asp Leu Ala Ile Thr Ile 740 745 750 Cys Ile Val Leu Asn ThrLeu Phe Met Ala Met Glu His His Pro Met 755 760 765 Thr Glu Glu Phe LysAsn Val Leu Ala Xaa Gly Asn Leu Xaa Phe Thr 770 775 780 Gly Ile Phe AlaAla Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro 785 790 795 800 Tyr GluTyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile Val 805 810 815 ThrLeu Ser Leu Xaa Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser 820 825 830Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser 835 840845 Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly 850855 860 Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe865 870 875 880 Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys GluCys Val 885 890 895 Cys Lys Ile Asn Xaa Asp Cys Xaa Leu Pro Arg Trp HisMet Asn Asp 900 905 910 Phe Phe His Ser Phe Leu Ile Val Phe Arg Val LeuCys Gly Glu Trp 915 920 925 Ile Glu Thr Met Trp Asp Cys Met Glu Val AlaGly Gln Xaa Met Cys 930 935 940 Leu Ile Val Tyr Met Met Val Met Val IleGly Asn Leu Val Val Leu 945 950 955 960 Asn Leu Phe Leu Ala Leu Leu LeuSer Ser Phe Ser Ser Asp Asn Leu 965 970 975 Thr Ala Ile Glu Glu Asp XaaAsp Ala Asn Asn Leu Gln Ile Ala Val 980 985 990 Xaa Arg Ile Lys Xaa GlyIle Asn Tyr Val Lys Gln Thr Leu Arg Glu 995 1000 1005 Phe Ile Leu LysXaa Phe Ser Lys Lys Pro Lys Xaa Ser Xaa Xaa Xaa 1010 1015 1020 Xaa XaaXaa Xaa Asp Xaa Asn Xaa Lys Lys Glu Asn Tyr Ile Ser Asn 1025 1030 10351040 Xaa Thr Leu Ala Glu Met Ser Lys Xaa His Asn Phe Leu Lys Glu Lys1045 1050 1055 Asp Xaa Ile Ser Gly Xaa Gly Ser Ser Xaa Asp Lys Xaa XaaMet Xaa 1060 1065 1070 Xaa Xaa Asp Xaa Gln Ser Phe Ile His Asn Pro SerLeu Thr Val Thr 1075 1080 1085 Val Pro Ile Ala Pro Gly Glu Ser Asp LeuGlu Xaa Met Asn Xaa Glu 1090 1095 1100 Glu Leu Ser Ser Asp Ser Asp SerXaa Tyr Ser Lys Xaa Xaa Xaa Asn 1105 1110 1115 1120 Arg Ser Ser Ser SerGlu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly 1125 1130 1135 Glu Gly GluGlu Ala Glu Ala Glu Pro Xaa Asn Xaa Asp Glu Pro Glu 1140 1145 1150 AlaCys Phe Thr Asp Gly Cys Val Arg Arg Phe Xaa Cys Cys Gln Val 1155 11601165 Asn Xaa Xaa Ser Gly Lys Gly Lys Xaa Trp Trp Xaa Ile Arg Lys Thr1170 1175 1180 Cys Tyr Xaa Ile Val Glu His Ser Trp Phe Glu Ser Phe IleVal Leu 1185 1190 1195 1200 Met Ile Leu Leu Ser Ser Gly Ala Leu Ala PheGlu Asp Ile Tyr Ile 1205 1210 1215 Glu Xaa Lys Lys Thr Ile Lys Ile IleLeu Glu Tyr Ala Asp Lys Ile 1220 1225 1230 Phe Thr Tyr Ile Phe Ile LeuGlu Met Leu Leu Lys Trp Xaa Ala Tyr 1235 1240 1245 Gly Tyr Lys Thr TyrPhe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1250 1255 1260 Ile Val AspVal Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr 1265 1270 1275 1280Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg 12851290 1295 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val ValAsn 1300 1305 1310 Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn Val LeuLeu Val Cys 1315 1320 1325 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met GlyVal Asn Leu Phe Ala 1330 1335 1340 Gly Lys Phe Tyr Glu Cys Xaa Asn ThrThr Asp Gly Ser Arg Phe Pro 1345 1350 1355 1360 Xaa Ser Gln Val Xaa AsnArg Ser Glu Cys Phe Ala Leu Met Asn Val 1365 1370 1375 Ser Xaa Asn ValArg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val 1380 1385 1390 Gly LeuGly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1395 1400 1405Xaa Xaa Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Xaa Xaa Gln 14101415 1420 Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val XaaPhe 1425 1430 1435 1440 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu PheIle Gly Val Ile 1445 1450 1455 Ile Asp Asn Phe Asn Gln Gln Lys Lys LysLeu Gly Gly Gln Asp Ile 1460 1465 1470 Phe Met Thr Glu Glu Gln Lys LysTyr Tyr Asn Ala Met Lys Lys Leu 1475 1480 1485 Gly Ser Lys Lys Pro GlnLys Pro Ile Pro Arg Pro Gly Asn Lys Xaa 1490 1495 1500 Gln Gly Cys IlePhe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Xaa 1505 1510 1515 1520 IleMet Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys 1525 15301535 Glu Gly Gln Xaa Xaa Xaa Met Xaa Xaa Val Leu Xaa Trp Ile Asn Xaa1540 1545 1550 Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val Leu Lys LeuIle Ser 1555 1560 1565 Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn IleXaa Xaa Phe Val 1570 1575 1580 Val Val Ile Xaa Ser Ile Val Gly Met PheLeu Ala Xaa Xaa Ile Glu 1585 1590 1595 1600 Xaa Tyr Phe Val Ser Pro ThrLeu Phe Arg Val Ile Arg Leu Ala Arg 1605 1610 1615 Ile Gly Arg Ile LeuArg Leu Xaa Lys Gly Ala Lys Gly Ile Arg Thr 1620 1625 1630 Leu Leu PheAla Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1635 1640 1645 LeuLeu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 1650 16551660 Asn Phe Ala Tyr Val Lys Lys Glu Xaa Gly Ile Asn Asp Met Phe Asn1665 1670 1675 1680 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe GlnIle Thr Thr 1685 1690 1695 Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro IleLeu Asn Ser Xaa Pro 1700 1705 1710 Pro Asp Cys Asp Pro Lys Lys Val HisPro Gly Ser Ser Val Glu Gly 1715 1720 1725 Asp Cys Gly Asn Pro Ser ValGly Ile Phe Tyr Phe Val Ser Tyr Ile 1730 1735 1740 Ile Ile Ser Phe LeuVal Val Val Asn Met Tyr Ile Ala Val Ile Leu 1745 1750 1755 1760 Glu AsnPhe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu 1765 1770 1775Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 17801785 1790 Ala Thr Gln Phe Ile Glu Phe Xaa Lys Leu Ser Asp Phe Ala AlaAla 1795 1800 1805 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys ValGln Leu Ile 1810 1815 1820 Ala Met Asp Leu Pro Met Val Ser Gly Asp ArgIle His Cys Leu Asp 1825 1830 1835 1840 Ile Leu Phe Ala Phe Thr Lys ArgVal Leu Gly Glu Xaa Gly Glu Met 1845 1850 1855 Asp Ser Leu Arg Ser GlnMet Glu Glu Arg Phe Met Ser Ala Asn Pro 1860 1865 1870 Ser Lys Val SerTyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1875 1880 1885 Glu XaaVal Ser Ala Thr Xaa Ile Gln Arg Ala Tyr Arg Arg Tyr Arg 1890 1895 1900Leu Arg Gln Xaa Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly 19051910 1915 1920 Asp Arg Asp Asp Asp Leu Xaa Asn Lys Xaa Asp Xaa Xaa PheAsp Asn 1925 1930 1935 Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp XaaThr Xaa Ser Thr 1940 1945 1950 Xaa Ser Pro Pro Ser Tyr Asp Ser Val ThrLys Pro Asp Xaa Glu Lys 1955 1960 1965 Tyr Glu Xaa Asp Xaa Thr Glu LysGlu Asp Lys Xaa Lys Asp Ser Lys 1970 1975 1980 Glu Ser Xaa Lys Xaa 19851989 amino acids amino acid Not Relevant Not Relevant protein 12 Met AlaMet Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 LysGln Ser Leu Ala Leu Ile Glu Gln Arg Ile Ser Glu Glu Lys Ala 20 25 30 LysGlu His Lys Asp Glu Lys Lys Asp Asp Glu Glu Glu Gly Pro Lys 35 40 45 ProSer Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 AspIle Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Ala 85 90 95Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr130 135 140 Leu Ser Asn Pro Pro Glu Trp Thr Lys Asn Val Gly Tyr Thr PheThr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Leu AlaArg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro TrpAsn Trp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr GluPhe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg ValLeu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys ThrIle Val Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser AspVal Met Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu IleGly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe ArgLys Glu Leu Glu Glu Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn ThrAla Glu Ser Glu Glu Glu Leu Lys Lys Tyr Phe Tyr 290 295 300 Tyr Leu GluGly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 SerGly Gln Cys Pro Glu Gly Tyr Ile Cys Val Lys Ala Gly Arg Asn 325 330 335Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu AlaVal 385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn IleGlu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu AspArg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala AlaAla Ala Glu Phe Thr 435 440 445 Ser Ile Arg Arg Ser Arg Ile Met Gly LeuSer Glu Ser Ser Ser Glu 450 455 460 Thr Ser Arg Leu Ser Ser Lys Ser AlaLys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Lys Gln Lys XaaMet Ser Ser Gly Glu Glu Lys Gly Asp 485 490 495 Asp Glu Lys Leu Ser LysSer Gly Ser Glu Glu Ser Ile Arg Lys Lys 500 505 510 Ser Phe His Leu GlyVal Glu Gly His His Arg Thr Arg Glu Lys Arg 515 520 525 Leu Ser Thr ProAsn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala ArgArg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 GlyArg Asp Leu Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585590 Arg Pro Arg Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595600 605 Pro Pro Val Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys610 615 620 Asn Gly Val Val Ser Leu Val Asp Gly Pro Ser Ala Leu Met LeuPro 625 630 635 640 Asn Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys AlaThr Ser Asp 645 650 655 Asp Ser Gly Thr Thr Asn Gln Met Arg Lys Lys ArgLeu Ser Ser Ser 660 665 670 Tyr Phe Leu Ser Glu Asp Met Leu Asn Asp ProHis Leu Arg Gln Arg 675 680 685 Ala Met Ser Arg Ala Ser Ile Leu Thr AsnThr Val Glu Glu Leu Glu 690 695 700 Glu Ser Arg Gln Lys Cys His Gln LeuLeu Tyr Arg Phe Ala His Thr 705 710 715 720 Phe Leu Ile Trp Asn Cys SerPro Tyr Trp Ile Lys Phe Lys Lys Leu 725 730 735 Ile Tyr Phe Ile Val MetAsp Pro Phe Val Asp Leu Ala Ile Thr Ile 740 745 750 Cys Ile Val Leu AsnThr Leu Phe Met Ala Met Glu His His Pro Met 755 760 765 Thr Glu Glu PheLys Asn Val Leu Ala Val Gly Asn Leu Ile Phe Thr 770 775 780 Gly Ile PheAla Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro 785 790 795 800 TyrGlu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile Val 805 810 815Thr Leu Ser Leu Ile Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser 820 825830 Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser 835840 845 Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly850 855 860 Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe IlePhe 865 870 875 880 Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr LysGlu Cys Val 885 890 895 Cys Lys Ile Asn Val Asp Cys Lys Leu Pro Arg TrpHis Met Asn Asp 900 905 910 Phe Phe His Ser Phe Leu Ile Val Phe Arg ValLeu Cys Gly Glu Trp 915 920 925 Ile Glu Thr Met Trp Asp Cys Met Glu ValAla Gly Gln Thr Met Cys 930 935 940 Leu Ile Val Tyr Met Met Val Met ValIle Gly Asn Leu Val Val Leu 945 950 955 960 Asn Leu Phe Leu Ala Leu LeuLeu Ser Ser Phe Ser Ser Asp Asn Leu 965 970 975 Thr Ala Ile Glu Glu AspThr Asp Ala Asn Asn Leu Gln Ile Ala Val 980 985 990 Ala Arg Ile Lys ArgGly Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu 995 1000 1005 Phe Ile LeuLys Ser Phe Ser Lys Lys Pro Lys Gly Ser Lys Asp Thr 1010 1015 1020 LysArg Thr Ala Asp Pro Asn Asn Lys Lys Glu Asn Tyr Ile Ser Asn 1025 10301035 1040 Arg Thr Leu Ala Glu Met Ser Lys Asp His Asn Phe Leu Lys GluLys 1045 1050 1055 Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu Asp Lys SerPhe Met Asp 1060 1065 1070 Glu Asn Asp Tyr Gln Ser Phe Ile His Asn ProSer Leu Thr Val Thr 1075 1080 1085 Val Pro Ile Ala Pro Gly Glu Ser AspLeu Glu Ile Met Asn Thr Glu 1090 1095 1100 Glu Leu Ser Ser Asp Ser AspSer Asp Tyr Ser Lys Glu Lys Arg Asn 1105 1110 1115 1120 Arg Ser Ser SerSer Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly 1125 1130 1135 Glu XaaGlu Glu Ala Glu Ala Glu Pro Val Asn Ala Asp Glu Pro Glu 1140 1145 1150Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe Pro Cys Cys Gln Val 11551160 1165 Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp Thr Ile Arg LysThr 1170 1175 1180 Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu Ser PheIle Val Leu 1185 1190 1195 1200 Met Ile Leu Leu Ser Ser Gly Ala Leu AlaPhe Glu Asp Ile Tyr Ile 1205 1210 1215 Glu Lys Lys Lys Thr Ile Lys IleIle Leu Glu Tyr Ala Asp Lys Ile 1220 1225 1230 Phe Thr Tyr Ile Phe IleLeu Glu Met Leu Leu Lys Trp Val Ala Tyr 1235 1240 1245 Gly Tyr Lys ThrTyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1250 1255 1260 Ile ValAsp Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr 1265 1270 12751280 Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg1285 1290 1295 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val ValVal Asn 1300 1305 1310 Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn ValLeu Leu Val Cys 1315 1320 1325 Leu Ile Phe Trp Leu Ile Phe Ser Ile MetGly Val Asn Leu Phe Ala 1330 1335 1340 Gly Lys Phe Tyr Glu Cys Val AsnThr Thr Asp Gly Ser Arg Phe Pro 1345 1350 1355 1360 Thr Ser Gln Val AlaAsn Arg Ser Glu Cys Phe Ala Leu Met Asn Val 1365 1370 1375 Ser Gly AsnVal Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val 1380 1385 1390 GlyLeu Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1395 14001405 Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Asn Glu Gln1410 1415 1420 Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe ValIle Phe 1425 1430 1435 1440 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn LeuPhe Ile Gly Val Ile 1445 1450 1455 Ile Asp Asn Phe Asn Gln Gln Lys LysLys Leu Gly Gly Gln Asp Ile 1460 1465 1470 Phe Met Thr Glu Glu Gln LysLys Tyr Tyr Asn Ala Met Lys Lys Leu 1475 1480 1485 Gly Ser Lys Lys ProGln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe 1490 1495 1500 Gln Gly CysIle Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Thr 1505 1510 1515 1520Ile Met Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys 15251530 1535 Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu His Trp Ile AsnMet 1540 1545 1550 Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val Leu LysLeu Ile Ser 1555 1560 1565 Leu Arg His Tyr Tyr Phe Thr Val Gly Trp AsnIle Leu Tyr Phe Val 1570 1575 1580 Val Val Ile Leu Ser Ile Val Gly MetPhe Leu Ala Glu Met Ile Glu 1585 1590 1595 1600 Lys Tyr Phe Val Ser ProThr Leu Phe Arg Val Ile Arg Leu Ala Arg 1605 1610 1615 Ile Gly Arg IleLeu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr 1620 1625 1630 Leu LeuPhe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1635 1640 1645Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 16501655 1660 Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile Asn Asp Met PheAsn 1665 1670 1675 1680 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu PheGln Ile Thr Thr 1685 1690 1695 Ser Ala Gly Trp Asp Gly Leu Leu Ala ProIle Leu Asn Ser Ala Pro 1700 1705 1710 Pro Asp Cys Asp Pro Lys Lys ValHis Pro Gly Ser Ser Val Glu Gly 1715 1720 1725 Asp Cys Gly Asn Pro SerVal Gly Ile Phe Tyr Phe Val Ser Tyr Ile 1730 1735 1740 Ile Ile Ser PheLeu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu 1745 1750 1755 1760 GluAsn Phe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu 1765 17701775 Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp1780 1785 1790 Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe AlaAla Ala 1795 1800 1805 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn LysVal Gln Leu Ile 1810 1815 1820 Ala Met Asp Leu Pro Met Val Ser Gly AspArg Ile His Cys Leu Asp 1825 1830 1835 1840 Ile Leu Phe Ala Phe Thr LysArg Val Leu Gly Glu Gly Gly Glu Met 1845 1850 1855 Asp Ser Leu Arg SerGln Met Glu Glu Arg Phe Met Ser Ala Asn Pro 1860 1865 1870 Ser Lys ValSer Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1875 1880 1885 GluGlu Val Ser Ala Thr Ile Ile Gln Arg Ala Tyr Arg Arg Tyr Arg 1890 18951900 Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly1905 1910 1915 1920 Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp Thr ValPhe Asp Asn 1925 1930 1935 Val Asn Glu Asn Ser Ser Pro Glu Lys Thr AspVal Thr Ala Ser Thr 1940 1945 1950 Ile Ser Pro Pro Ser Tyr Asp Ser ValThr Lys Pro Asp Gln Glu Lys 1955 1960 1965 Tyr Glu Thr Asp Lys Thr GluLys Glu Asp Lys Glu Lys Asp Xaa Xaa 1970 1975 1980 Glu Ser Arg Lys Xaa1985 6371 base pairs nucleic acid both both DNA (genomic) 13 CTCTTATGTGAGGAGCTGAA GAGGAATTAA AATATACAGG ATGAAAAGAT GGCAATGTTG 60 CCTCCCCCAGGACCTCAGAG CTTTGTCCAT TTCACAAAAC AGTCTCTTGC CCTCATTGAA 120 CAACGCATTGCTGAAAGAAA ATCAAAGGAA CCCAAAGAAG AAAAGAAAGA TGATGATGAA 180 GAAGCCCCAAAGCCAAGCAG TGACTTGGAA GCTGGCAAAC AACTGCCCTT CATCTATGGG 240 GACATTCCTCCCGGCATGGT GTCAGAGCCC CTGGAGGACT TGGACCCCTA CTATGCAGAC 300 AAAAAGACTTTCATAGTATT GAACAAAGGG AAAACAATCT TCCGTTTCAA TGCCACACCT 360 GCTTTATATATGCTTTCTCC TTTCAGTCCT CTAAGAAGAA TATCTATTAA GATTTTAGTA 420 CACTCCTTATTCAGCATGCT CATCATGTGC ACTATTCTGA CAAACTGCAT ATTTATGACC 480 ATGAATAACCCGCCGGACTG GACCAAAAAT GTCGAGTACA CTTTTACTGG AATATATACT 540 TTTGAATCACTTGTAAAAAT CCTTGCAAGA GGCTTCTGTG TAGGAGAATT CACTTTTCTT 600 CGTGACCCGTGGAACTGGCT GGATTTTGTC GTCATTGTTT TTGCGTATTT AACAGAATTT 660 GTAAACCTAGGCAATGTTTC AGCTCTTCGA ACTTTCAGAG TATTGAGAGC TTTGAAAACT 720 ATTTCTGTAATCCCAGGCCT GAAGACAATT GTAGGGGCTT TGATCCAGTC AGTGAAGAAG 780 CTTTCTGATGTCATGATCCT GACTGTGTTC TGTCTGAGTG TGTTTGCACT AATTGGACTA 840 CAGCTGTTCATGGGAAACCT GAAGCATAAA TGTTTTCGAA ATTCACTTGA AAATAATGAA 900 ACATTAGAAAGCATAATGAA TACCCTAGAG AGTGAAGAAG ACTTTAGAAA ATATTTTTAT 960 TACTTGGAAGGATCCAAAGA TGCTCTCCTT TGTGGTTTCA GCACAGATTC AGGTCAGTGT 1020 CCAGAGGGGTACACCTGTGT GAAAATTGGC AGAAACCCTG ATTATGGCTA CACGAGCTTT 1080 GACACTTTCAGCTGGGCCTT CTTAGCCTTG TTTAGGCTAA TGACCCAAGA TTACTGGGAA 1140 AACCTTTACCAACAGACGCT GCGTGCTGCT GGCAAAACCT ACATGATCTT CTTTGTCGTA 1200 GTGATTTTCCTGGGCTCCTT TTATCTAATA AACTTGATCC TGGCTGTGGT TGCCATGGCA 1260 TATGAAGAACAGAACCAGGC AAACATTGAA GAAGCTAAAC AGAAAGAATT AGAATTTCAA 1320 CAGATGTTAGACCGTCTTAA AAAAGAGCAA GAAGAAGCTG AGGCAATTGC AGCGGCAGCG 1380 GCTGAATATACAAGTATTAG GAGAAGCAGA ATTATGGGCC TCTCAGAGAG TTCTTCTGAA 1440 ACATCCAAACTGAGCTCTAA AAGTGCTAAA GAAAGAAGAA ACAGAAGAAA GAAAAAGAAT 1500 CAAAAGAAGCTCTCCAGTGG AGAGGAAAAG GGAGATGCTG AGAAATTGTC GAAATCAGAA 1560 TCAGAGGACAGCATCAGAAG AAAAAGTTTC CACCTTGGTG TCGAAGGGCA TAGGCGAGCA 1620 CATGAAAAGAGGTTGTCTAC CCCCAATCAG TCACCACTCA GCATTCGTGG CTCCTTGTTT 1680 TCTGCAAGGCGAAGCAGCAG AACAAGTCTT TTTAGTTTCA AAGGCAGAGG AAGAGATATA 1740 GGATCTGAGACTGAATTTGC CGATGATGAG CACAGCATTT TTGGAGACAA TGAGAGCAGA 1800 AGGGGCTCACTGTTTGTGCC CCACAGACCC CAGGAGCGAC GCAGCAGTAA CATCAGCCAA 1860 GCCAGTAGGTCCCCACCAAT GCTGCCGGTG AACGGGAAAA TGCACAGTGC TGTGGACTGC 1920 AACGGTGTGGTCTCCCTGGT TGATGGACGC TCAGCCCTCA TGCTCCCCAA TGGACAGCTT 1980 CTGCCAGAGGGCACGACCAA TCAAATACAC AAGAAAAGGC GTTGTAGTTC CTATCTCCTT 2040 TCAGAGGATATGCTGAATGA TCCCAACCTC AGACAGAGAG CAATGAGTAG AGCAAGCATA 2100 TTAACAAACACTGTGGAAGA ACTTGAAGAG TCCAGACAAA AATGTCCACC TTGGTGGTAC 2160 AGATTTGCACACAAATTCTT GATCTGGAAT TGCTCTCCAT ATTGGATAAA ATTCAAAAAG 2220 TGTATCTATTTTATTGTAAT GGATCCTTTT GTAGATCTTG CAATTACCAT TTGCATAGTT 2280 TTAAACACATTATTTATGGC TATGGAACAC CACCCAATGA CTGAGGAATT CAAAAATGTA 2340 CTTGCTATAGGAAATTTGGT CTTTACTGGA ATCTTTGCAG CTGAAATGGT ATTAAAACTG 2400 ATTGCCATGGATCCATATGA GTATTTCCAA GTAGGCTGGA ATATTTTTGA CAGCCTTATT 2460 GTGACTTTAAGTTTAGTGGA GCTCTTTCTA GCAGATGTGG AAGGATTGTC AGTTCTGCGA 2520 TCATTCAGACTGCTCCGAGT CTTCAAGTTG GCAAAATCCT GGCCAACATT GAACATGCTG 2580 ATTAAGATCATTGGTAACTC AGTAGGGGCT CTAGGTAACC TCACCTTAGT GTTGGCCATC 2640 ATCGTCTTCATTTTTGCTGT GGTCGGCATG CAGCTCTTTG GTAAGAGCTA CAAAGAATGT 2700 GTCTGCAAGATCAATGATGA CTGTACGCTC CCACGGTGGC ACATGAACGA CTTCTTCCAC 2760 TCCTTCCTGATTGTGTTCCG CGTGCTGTGT GGAGAGTGGA TAGAGACCAT GTGGGACTGT 2820 ATGGAGGTCGCTGGTCAAGC TATGTGCCTT ATTGTTTACA TGATGGTCAT GGTCATTGGA 2880 AACCTGGTGGTCCTAAACCT ATTTCTGGCC TTATTATTGA GCTCATTTAG TTCAGACAAT 2940 CTTACAGCAATTGAAGAAGA CCCTGATGCA AACAACCTCC AGATTGCAGT GACTAGAATT 3000 AAAAAGGGAATAAATTATGT GAAACAAACC TTACGTGAAT TTATTCTAAA AGCATTTTCC 3060 AAAAAGCCAAAGATTTCCAG GGAGATAAGA CAAGCAGAAG ATCTGAATAC TAAGAAGGAA 3120 AACTATATTTCTAACCATAC ACTTGCTGAA ATGAGCAAAG GTCACAATTT CCTCAAGGAA 3180 AAAGATAAAATCAGTGGTTT TGGAAGCAGC GTGGACAAAC ACTTGATGGA AGACAGTGAT 3240 GGTCAATCATTTATTCACAA TCCCAGCCTC ACAGTGACAG TGCCAATTGC ACCTGGGGAA 3300 TCCGATTTGGAAAATATGAA TGCTGAGGAA CTTAGCAGTG ATTCGGATAG TGAATACAGC 3360 AAAGTGAGATTAAACCGGTC AAGCTCCTCA GAGTGCAGCA CAGTTGATAA CCCTTTGCCT 3420 GGAGAAGGAGAAGAAGCAGA GGCTGAACCT ATGAATTCCG ATGAGCCAGA GGCCTGTTTC 3480 ACAGATGGTTGTGTACGGAG GTTCTCATGC TGCCAAGTTA ACATAGAGTC AGGGAAAGGA 3540 AAAATCTGGTGGAACATCAG GAAAACCTGC TACAAGATTG TTGAACACAG TTGGTTTGAA 3600 AGCTTCATTGTCCTCATGAT CCTGCTCAGC AGTGGTGCCC TGGCTTTTGA AGATATTTAT 3660 ATTGAAAGGAAAAAGACCAT TAAGATTATC CTGGAGTATG CAGACAAGAT CTTCACTTAC 3720 ATCTTCATTCTGGAAATGCT TCTAAAATGG ATAGCATATG GTTATAAAAC ATATTTCACC 3780 AATGCCTGGTGTTGGCTGGA TTTCCTAATT GTTGATGTTT CTTTGGTTAC TTTAGTGGCA 3840 AACACTCTTGGCTACTCAGA TCTTGGCCCC ATTAAATCCC TTCGGACACT GAGAGCTTTA 3900 AGACCTCTAAGAGCCTTATC TAGATTTGAA GGAATGAGGG TCGTTGTGAA TGCACTCATA 3960 GGAGCAATTCCTTCCATCAT GAATGTGCTA CTTGTGTGTC TTATATTCTG GCTGATATTC 4020 AGCATCATGGGAGTAAATTT GTTTGCTGGC AAGTTCTATG AGTGTATTAA CACCACAGAT 4080 GGGTCACGGTTTCCTGCAAG TCAAGTTCCA AATCGTTCCG AATGTTTTGC CCTTATGAAT 4140 GTTAGTCAAAATGTGCGATG GAAAAACCTG AAAGTGAACT TTGATAATGT CGGACTTGGT 4200 TACCTATCTCTGCTTCAAGT TGCAACTTTT AAGGGATGGA CGATTATTAT GTATGCAGCA 4260 GTGGATTCTGTTAATGTAGA CAAGCAGCCC AAATATGAAT ATAGCCTCTA CATGTATATT 4320 TATTTTGTCGTCTTTATCAT CTTTGGGTCA TTCTTCACTT TGAACTTGTT CATTGGTGTC 4380 ATCATAGATAATTTCAACCA ACAGAAAAAG AAGCTTGGAG GTCAAGACAT CTTTATGACA 4440 GAAGAACAGAAGAAATACTA TAATGCAATG AAAAAGCTGG GGTCCAAGAA GCCACAAAAG 4500 CCAATTCCTCGACCAGGGAA CAAAATCCAA GGATGTATAT TTGACCTAGT GACAAATCAA 4560 GCCTTTGATATTAGTATCAT GGTTCTTATC TGTCTCAACA TGGTAACCAT GATGGTAGAA 4620 AAGGAGGGTCAAAGTCAACA TATGACTGAA GTTTTATATT GGATAAATGT GGTTTTTATA 4680 ATCCTTTTCACTGGAGAATG TGTGCTAAAA CTGATCTCCC TCAGACACTA CTACTTCACT 4740 GTAGGATGGAATATTTTTGA TTTTGTGGTT GTGATTATCT CCATTGTAGG TATGTTTCTA 4800 GCTGATTTGATTGAAACGTA TTTTGTGTCC CCTACCCTGT TCCGAGTGAT CCGTCTTGCC 4860 AGGATTGGCCGAATCCTACG TCTAGTCAAA GGAGCAAAGG GGATCCGCAC GCTGCTCTTT 4920 GCTTTGATGATGTCCCTTCC TGCGTTGTTT AACATCGGCC TCCTGCTCTT CCTGGTCATG 4980 TTCATCTACGCCATCTTTGG AATGTCCAAC TTTGCCTATG TTAAAAAGGA AGATGGAATT 5040 AATGACATGTTCAATTTTGA GACCTTTGGC AACAGTATGA TTTGCCTGTT CCAAATTACA 5100 ACCTCTGCTGGCTGGGATGG ATTGCTAGCA CCTATTCTTA ACAGTAAGCC ACCCGACTGT 5160 GACCCAAAAAAAGTTCATCC TGGAAGTTCA GTTGAAGGAG ACTGTGGTAA CCCATCTGTT 5220 GGAATATTCTACTTTGTTAG TTATATCATC ATATCCTTCC TGGTTGTGGT GAACATGTAC 5280 ATTGCAGTCATACTGGAGAA TTTTAGTGTT GCCACTGAAG AAAGTACTGA ACCTCTGAGT 5340 GAGGATGACTTTGAGATGTT CTATGAGGTT TGGGAGAAGT TTGATCCCGA TGCGACCCAG 5400 TTTATAGAGTTCTCTAAACT CTCTGATTTT GCAGCTGCCC TGGATCCTCC TCTTCTCATA 5460 GCAAAACCCAACAAAGTCCA GCTCATTGCC ATGGATCTGC CCATGGTTAG TGGTGACCGG 5520 ATCCATTGTCTTGACATCTT ATTTGCTTTT ACAAAGCGTG TTTTGGGTGA GAGTGGGGAG 5580 ATGGATTCTCTTCGTTCACA GATGGAAGAA AGGTTCATGT CTGCAAATCC TTCCAAAGTG 5640 TCCTATGAACCCATCACAAC CACACTAAAA CGGAAACAAG AGGATGTGTC TGCTACTGTC 5700 ATTCAGCGTGCTTATAGACG TTACCGCTTA AGGCAAAATG TCAAAAATAT ATCAAGTATA 5760 TACATAAAAGATGGAGACAG AGATGATGAT TTACTCAATA AAAAAGATAT GGCTTTTGAT 5820 AATGTTAATGAGAACTCAAG TCCAGAAAAA ACAGATGCCA CTTCATCCAC CACCTCTCCA 5880 CCTTCATATGATAGTGTAAC AAAGCCAGAC AAAGAGAAAT ATGAACAAGA CAGAACAGAA 5940 AAGGAAGACAAAGGGAAAGA CAGCAAGGAA AGCAAAAAAT AGAGCTTCAT TTTTGATATA 6000 TTGTTTACAGCCTGTGAAAG TGATTTATTT GTGTTAATAA AACTCTTTTG AGGAAGTCTA 6060 TGCCAAAATCCTTTTTATCA AAATATTCTC GAAGGCAGTG CAGTCACTAA CTCTGATTTC 6120 CTAAGAAAGGTGGGCAGCAT TAGCAGATGG TTATTTTTGC ACTGATGATT CTTTAAGAAT 6180 CGTAAGAGAACTCTGTAGGA ATTATTGATT ATAGCATACA AAAGTGATTG ATTCAGTTTT 6240 TTGGTTTTTAATAAATCAGA AGACCATGTA GAAAACTTTT ACATCTGCCT TGTCATCTTT 6300 TCACAGGATTGTAATTAGTC TTGTTTCCCA TGTAAATAAA CAACACACGC ATACAGAAAA 6360 AAAAAAAAAA A6371 6404 base pairs nucleic acid both both DNA (genomic) 14 CTCTTATGTGAGGAGCTGAA GAGGAATTAA AATATACAGG ATGAAAAGAT GGCAATGTTG 60 CCTCCCCCAGGACCTCAGAG CTTTGTCCAT TTCACAAAAC AGTCTCTTGC CCTCATTGAA 120 CAACGCATTGCTGAAAGAAA ATCAAAGGAA CCCAAAGAAG AAAAGAAAGA TGATGATGAA 180 GAAGCCCCAAAGCCAAGCAG TGACTTGGAA GCTGGCAAAC AACTGCCCTT CATCTATGGG 240 GACATTCCTCCCGGCATGGT GTCAGAGCCC CTGGAGGACT TGGACCCCTA CTATGCAGAC 300 AAAAAGACTTTCATAGTATT GAACAAAGGG AAAACAATCT TCCGTTTCAA TGCCACACCT 360 GCTTTATATATGCTTTCTCC TTTCAGTCCT CTAAGAAGAA TATCTATTAA GATTTTAGTA 420 CACTCCTTATTCAGCATGCT CATCATGTGC ACTATTCTGA CAAACTGCAT ATTTATGACC 480 ATGAATAACCCGCCGGACTG GACCAAAAAT GTCGAGTACA CTTTTACTGG AATATATACT 540 TTTGAATCACTTGTAAAAAT CCTTGCAAGA GGCTTCTGTG TAGGAGAATT CACTTTTCTT 600 CGTGACCCGTGGAACTGGCT GGATTTTGTC GTCATTGTTT TTGCGTATTT AACAGAATTT 660 GTAAACCTAGGCAATGTTTC AGCTCTTCGA ACTTTCAGAG TATTGAGAGC TTTGAAAACT 720 ATTTCTGTAATCCCAGGCCT GAAGACAATT GTAGGGGCTT TGATCCAGTC AGTGAAGAAG 780 CTTTCTGATGTCATGATCCT GACTGTGTTC TGTCTGAGTG TGTTTGCACT AATTGGACTA 840 CAGCTGTTCATGGGAAACCT GAAGCATAAA TGTTTTCGAA ATTCACTTGA AAATAATGAA 900 ACATTAGAAAGCATAATGAA TACCCTAGAG AGTGAAGAAG ACTTTAGAAA ATATTTTTAT 960 TACTTGGAAGGATCCAAAGA TGCTCTCCTT TGTGGTTTCA GCACAGATTC AGGTCAGTGT 1020 CCAGAGGGGTACACCTGTGT GAAAATTGGC AGAAACCCTG ATTATGGCTA CACGAGCTTT 1080 GACACTTTCAGCTGGGCCTT CTTAGCCTTG TTTAGGCTAA TGACCCAAGA TTACTGGGAA 1140 AACCTTTACCAACAGACGCT GCGTGCTGCT GGCAAAACCT ACATGATCTT CTTTGTCGTA 1200 GTGATTTTCCTGGGCTCCTT TTATCTAATA AACTTGATCC TGGCTGTGGT TGCCATGGCA 1260 TATGAAGAACAGAACCAGGC AAACATTGAA GAAGCTAAAC AGAAAGAATT AGAATTTCAA 1320 CAGATGTTAGACCGTCTTAA AAAAGAGCAA GAAGAAGCTG AGGCAATTGC AGCGGCAGCG 1380 GCTGAATATACAAGTATTAG GAGAAGCAGA ATTATGGGCC TCTCAGAGAG TTCTTCTGAA 1440 ACATCCAAACTGAGCTCTAA AAGTGCTAAA GAAAGAAGAA ACAGAAGAAA GAAAAAGAAT 1500 CAAAAGAAGCTCTCCAGTGG AGAGGAAAAG GGAGATGCTG AGAAATTGTC GAAATCAGAA 1560 TCAGAGGACAGCATCAGAAG AAAAAGTTTC CACCTTGGTG TCGAAGGGCA TAGGCGAGCA 1620 CATGAAAAGAGGTTGTCTAC CCCCAATCAG TCACCACTCA GCATTCGTGG CTCCTTGTTT 1680 TCTGCAAGGCGAAGCAGCAG AACAAGTCTT TTTAGTTTCA AAGGCAGAGG AAGAGATATA 1740 GGATCTGAGACTGAATTTGC CGATGATGAG CACAGCATTT TTGGAGACAA TGAGAGCAGA 1800 AGGGGCTCACTGTTTGTGCC CCACAGACCC CAGGAGCGAC GCAGCAGTAA CATCAGCCAA 1860 GCCAGTAGGTCCCCACCAAT GCTGCCGGTG AACGGGAAAA TGCACAGTGC TGTGGACTGC 1920 AACGGTGTGGTCTCCCTGGT TGATGGACGC TCAGCCCTCA TGCTCCCCAA TGGACAGCTT 1980 CTGCCAGAGGTGATAATAGA TAAGACAACT TCTGATGACA GCGGCACGAC CAATCAAATA 2040 CACAAGAAAAGGCGTTGTAG TTCCTATCTC CTTTCAGAGG ATATGCTGAA TGATCCCAAC 2100 CTCAGACAGAGAGCAATGAG TAGAGCAAGC ATATTAACAA ACACTGTGGA AGAACTTGAA 2160 GAGTCCAGACAAAAATGTCC ACCTTGGTGG TACAGATTTG CACACAAATT CTTGATCTGG 2220 AATTGCTCTCCATATTGGAT AAAATTCAAA AAGTGTATCT ATTTTATTGT AATGGATCCT 2280 TTTGTAGATCTTGCAATTAC CATTTGCATA GTTTTAAACA CATTATTTAT GGCTATGGAA 2340 CACCACCCAATGACTGAGGA ATTCAAAAAT GTACTTGCTA TAGGAAATTT GGTCTTTACT 2400 GGAATCTTTGCAGCTGAAAT GGTATTAAAA CTGATTGCCA TGGATCCATA TGAGTATTTC 2460 CAAGTAGGCTGGAATATTTT TGACAGCCTT ATTGTGACTT TAAGTTTAGT GGAGCTCTTT 2520 CTAGCAGATGTGGAAGGATT GTCAGTTCTG CGATCATTCA GACTGCTCCG AGTCTTCAAG 2580 TTGGCAAAATCCTGGCCAAC ATTGAACATG CTGATTAAGA TCATTGGTAA CTCAGTAGGG 2640 GCTCTAGGTAACCTCACCTT AGTGTTGGCC ATCATCGTCT TCATTTTTGC TGTGGTCGGC 2700 ATGCAGCTCTTTGGTAAGAG CTACAAAGAA TGTGTCTGCA AGATCAATGA TGACTGTACG 2760 CTCCCACGGTGGCACATGAA CGACTTCTTC CACTCCTTCC TGATTGTGTT CCGCGTGCTG 2820 TGTGGAGAGTGGATAGAGAC CATGTGGGAC TGTATGGAGG TCGCTGGTCA AGCTATGTGC 2880 CTTATTGTTTACATGATGGT CATGGTCATT GGAAACCTGG TGGTCCTAAA CCTATTTCTG 2940 GCCTTATTATTGAGCTCATT TAGTTCAGAC AATCTTACAG CAATTGAAGA AGACCCTGAT 3000 GCAAACAACCTCCAGATTGC AGTGACTAGA ATTAAAAAGG GAATAAATTA TGTGAAACAA 3060 ACCTTACGTGAATTTATTCT AAAAGCATTT TCCAAAAAGC CAAAGATTTC CAGGGAGATA 3120 AGACAAGCAGAAGATCTGAA TACTAAGAAG GAAAACTATA TTTCTAACCA TACACTTGCT 3180 GAAATGAGCAAAGGTCACAA TTTCCTCAAG GAAAAAGATA AAATCAGTGG TTTTGGAAGC 3240 AGCGTGGACAAACACTTGAT GGAAGACAGT GATGGTCAAT CATTTATTCA CAATCCCAGC 3300 CTCACAGTGACAGTGCCAAT TGCACCTGGG GAATCCGATT TGGAAAATAT GAATGCTGAG 3360 GAACTTAGCAGTGATTCGGA TAGTGAATAC AGCAAAGTGA GATTAAACCG GTCAAGCTCC 3420 TCAGAGTGCAGCACAGTTGA TAACCCTTTG CCTGGAGAAG GAGAAGAAGC AGAGGCTGAA 3480 CCTATGAATTCCGATGAGCC AGAGGCCTGT TTCACAGATG GTTGTGTACG GAGGTTCTCA 3540 TGCTGCCAAGTTAACATAGA GTCAGGGAAA GGAAAAATCT GGTGGAACAT CAGGAAAACC 3600 TGCTACAAGATTGTTGAACA CAGTTGGTTT GAAAGCTTCA TTGTCCTCAT GATCCTGCTC 3660 AGCAGTGGTGCCCTGGCTTT TGAAGATATT TATATTGAAA GGAAAAAGAC CATTAAGATT 3720 ATCCTGGAGTATGCAGACAA GATCTTCACT TACATCTTCA TTCTGGAAAT GCTTCTAAAA 3780 TGGATAGCATATGGTTATAA AACATATTTC ACCAATGCCT GGTGTTGGCT GGATTTCCTA 3840 ATTGTTGATGTTTCTTTGGT TACTTTAGTG GCAAACACTC TTGGCTACTC AGATCTTGGC 3900 CCCATTAAATCCCTTCGGAC ACTGAGAGCT TTAAGACCTC TAAGAGCCTT ATCTAGATTT 3960 GAAGGAATGAGGGTCGTTGT GAATGCACTC ATAGGAGCAA TTCCTTCCAT CATGAATGTG 4020 CTACTTGTGTGTCTTATATT CTGGCTGATA TTCAGCATCA TGGGAGTAAA TTTGTTTGCT 4080 GGCAAGTTCTATGAGTGTAT TAACACCACA GATGGGTCAC GGTTTCCTGC AAGTCAAGTT 4140 CCAAATCGTTCCGAATGTTT TGCCCTTATG AATGTTAGTC AAAATGTGCG ATGGAAAAAC 4200 CTGAAAGTGAACTTTGATAA TGTCGGACTT GGTTACCTAT CTCTGCTTCA AGTTGCAACT 4260 TTTAAGGGATGGACGATTAT TATGTATGCA GCAGTGGATT CTGTTAATGT AGACAAGCAG 4320 CCCAAATATGAATATAGCCT CTACATGTAT ATTTATTTTG TCGTCTTTAT CATCTTTGGG 4380 TCATTCTTCACTTTGAACTT GTTCATTGGT GTCATCATAG ATAATTTCAA CCAACAGAAA 4440 AAGAAGCTTGGAGGTCAAGA CATCTTTATG ACAGAAGAAC AGAAGAAATA CTATAATGCA 4500 ATGAAAAAGCTGGGGTCCAA GAAGCCACAA AAGCCAATTC CTCGACCAGG GAACAAAATC 4560 CAAGGATGTATATTTGACCT AGTGACAAAT CAAGCCTTTG ATATTAGTAT CATGGTTCTT 4620 ATCTGTCTCAACATGGTAAC CATGATGGTA GAAAAGGAGG GTCAAAGTCA ACATATGACT 4680 GAAGTTTTATATTGGATAAA TGTGGTTTTT ATAATCCTTT TCACTGGAGA ATGTGTGCTA 4740 AAACTGATCTCCCTCAGACA CTACTACTTC ACTGTAGGAT GGAATATTTT TGATTTTGTG 4800 GTTGTGATTATCTCCATTGT AGGTATGTTT CTAGCTGATT TGATTGAAAC GTATTTTGTG 4860 TCCCCTACCCTGTTCCGAGT GATCCGTCTT GCCAGGATTG GCCGAATCCT ACGTCTAGTC 4920 AAAGGAGCAAAGGGGATCCG CACGCTGCTC TTTGCTTTGA TGATGTCCCT TCCTGCGTTG 4980 TTTAACATCGGCCTCCTGCT CTTCCTGGTC ATGTTCATCT ACGCCATCTT TGGAATGTCC 5040 AACTTTGCCTATGTTAAAAA GGAAGATGGA ATTAATGACA TGTTCAATTT TGAGACCTTT 5100 GGCAACAGTATGATTTGCCT GTTCCAAATT ACAACCTCTG CTGGCTGGGA TGGATTGCTA 5160 GCACCTATTCTTAACAGTAA GCCACCCGAC TGTGACCCAA AAAAAGTTCA TCCTGGAAGT 5220 TCAGTTGAAGGAGACTGTGG TAACCCATCT GTTGGAATAT TCTACTTTGT TAGTTATATC 5280 ATCATATCCTTCCTGGTTGT GGTGAACATG TACATTGCAG TCATACTGGA GAATTTTAGT 5340 GTTGCCACTGAAGAAAGTAC TGAACCTCTG AGTGAGGATG ACTTTGAGAT GTTCTATGAG 5400 GTTTGGGAGAAGTTTGATCC CGATGCGACC CAGTTTATAG AGTTCTCTAA ACTCTCTGAT 5460 TTTGCAGCTGCCCTGGATCC TCCTCTTCTC ATAGCAAAAC CCAACAAAGT CCAGCTCATT 5520 GCCATGGATCTGCCCATGGT TAGTGGTGAC CGGATCCATT GTCTTGACAT CTTATTTGCT 5580 TTTACAAAGCGTGTTTTGGG TGAGAGTGGG GAGATGGATT CTCTTCGTTC ACAGATGGAA 5640 GAAAGGTTCATGTCTGCAAA TCCTTCCAAA GTGTCCTATG AACCCATCAC AACCACACTA 5700 AAACGGAAACAAGAGGATGT GTCTGCTACT GTCATTCAGC GTGCTTATAG ACGTTACCGC 5760 TTAAGGCAAAATGTCAAAAA TATATCAAGT ATATACATAA AAGATGGAGA CAGAGATGAT 5820 GATTTACTCAATAAAAAAGA TATGGCTTTT GATAATGTTA ATGAGAACTC AAGTCCAGAA 5880 AAAACAGATGCCACTTCATC CACCACCTCT CCACCTTCAT ATGATAGTGT AACAAAGCCA 5940 GACAAAGAGAAATATGAACA AGACAGAACA GAAAAGGAAG ACAAAGGGAA AGACAGCAAG 6000 GAAAGCAAAAAATAGAGCTT CATTTTTGAT ATATTGTTTA CAGCCTGTGA AAGTGATTTA 6060 TTTGTGTTAATAAAACTCTT TTGAGGAAGT CTATGCCAAA ATCCTTTTTA TCAAAATATT 6120 CTCGAAGGCAGTGCAGTCAC TAACTCTGAT TTCCTAAGAA AGGTGGGCAG CATTAGCAGA 6180 TGGTTATTTTTGCACTGATG ATTCTTTAAG AATCGTAAGA GAACTCTGTA GGAATTATTG 6240 ATTATAGCATACAAAAGTGA TTGATTCAGT TTTTTGGTTT TTAATAAATC AGAAGACCAT 6300 GTAGAAAACTTTTACATCTG CCTTGTCATC TTTTCACAGG ATTGTAATTA GTCTTGTTTC 6360 CCATGTAAATAAACAACACA CGCATACAGA AAAAAAAAAA AAAA 6404 1835 amino acids amino acidNot Relevant Not Relevant protein 15 Met Ala Met Leu Pro Pro Pro Gly ProGln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile GluGln Arg Ile Glu Lys Lys Glu Lys 20 25 30 Glu Lys Lys Asp Asp Glu Glu ProLys Pro Ser Ser Asp Leu Glu Ala 35 40 45 Gly Lys Gln Leu Pro Phe Ile TyrGly Asp Ile Pro Pro Gly Met Val 50 55 60 Ser Glu Pro Leu Glu Asp Leu AspPro Tyr Tyr Ala Asp Lys Lys Thr 65 70 75 80 Phe Ile Val Leu Asn Lys GlyLys Ile Phe Arg Phe Asn Ala Thr Pro 85 90 95 Ala Leu Tyr Met Leu Ser ProPhe Ser Pro Leu Arg Arg Ile Ser Ile 100 105 110 Lys Ile Leu Val His SerLeu Phe Ser Met Leu Ile Met Cys Thr Ile 115 120 125 Leu Thr Asn Cys IlePhe Met Thr Asn Pro Pro Trp Thr Lys Asn Val 130 135 140 Tyr Thr Phe ThrGly Ile Tyr Thr Phe Glu Ser Leu Lys Ile Leu Ala 145 150 155 160 Arg GlyPhe Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn 165 170 175 TrpLeu Asp Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val 180 185 190Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala 195 200205 Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala 210215 220 Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val225 230 235 240 Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu PheMet Gly 245 250 255 Asn Leu Lys His Lys Cys Phe Arg Leu Glu Asn Glu ThrLeu Glu Ser 260 265 270 Ile Met Asn Thr Glu Ser Glu Glu Lys Tyr Phe TyrTyr Leu Glu Gly 275 280 285 Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser ThrAsp Ser Gly Gln Cys 290 295 300 Pro Glu Gly Tyr Cys Val Lys Gly Arg AsnPro Asp Tyr Gly Tyr Thr 305 310 315 320 Ser Phe Asp Thr Phe Ser Trp AlaPhe Leu Ala Leu Phe Arg Leu Met 325 330 335 Thr Gln Asp Tyr Trp Glu AsnLeu Tyr Gln Gln Thr Leu Arg Ala Ala 340 345 350 Gly Lys Thr Tyr Met IlePhe Phe Val Val Val Ile Phe Leu Gly Ser 355 360 365 Phe Tyr Leu Ile AsnLeu Ile Leu Ala Val Val Ala Met Ala Tyr Glu 370 375 380 Glu Gln Asn GlnAla Asn Ile Glu Glu Ala Lys Gln Lys Glu Leu Glu 385 390 395 400 Phe GlnGln Met Leu Asp Arg Leu Lys Lys Glu Gln Glu Glu Ala Glu 405 410 415 AlaIle Ala Ala Ala Ala Ala Glu Thr Ser Ile Arg Ser Arg Ile Met 420 425 430Gly Leu Ser Glu Ser Ser Ser Glu Thr Ser Leu Ser Ser Lys Ser Ala 435 440445 Lys Glu Arg Arg Asn Arg Arg Lys Lys Lys Gln Lys Lys Ser Ser Gly 450455 460 Glu Glu Lys Gly Asp Glu Lys Leu Ser Lys Ser Ser Glu Ser Ile Arg465 470 475 480 Lys Ser Phe His Leu Gly Val Glu Gly His Arg Glu Lys ArgLeu Ser 485 490 495 Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser LeuPhe Ser Ala 500 505 510 Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe LysGly Arg Gly Arg 515 520 525 Asp Gly Ser Glu Thr Glu Phe Ala Asp Asp GluHis Ser Ile Phe Gly 530 535 540 Asp Asn Glu Ser Arg Arg Gly Ser Leu PheVal Pro His Arg Pro Glu 545 550 555 560 Arg Arg Ser Ser Asn Ile Ser GlnAla Ser Arg Ser Pro Pro Leu Pro 565 570 575 Val Asn Gly Lys Met His SerAla Val Asp Cys Asn Gly Val Val Ser 580 585 590 Leu Val Asp Gly Ser AlaLeu Met Leu Pro Asn Gly Gln Leu Leu Pro 595 600 605 Glu Gly Thr Thr AsnGln Lys Lys Arg Ser Ser Tyr Leu Ser Glu Asp 610 615 620 Met Leu Asn AspPro Leu Arg Gln Arg Ala Met Ser Arg Ala Ser Ile 625 630 635 640 Leu ThrAsn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Tyr 645 650 655 ArgPhe Ala His Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile Lys 660 665 670Phe Lys Lys Ile Tyr Phe Ile Val Met Asp Pro Phe Val Asp Leu Ala 675 680685 Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His 690695 700 His Pro Met Thr Glu Glu Phe Lys Asn Val Leu Ala Gly Asn Leu Phe705 710 715 720 Thr Gly Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile AlaMet Asp 725 730 735 Pro Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe AspSer Leu Ile 740 745 750 Val Thr Leu Ser Leu Glu Leu Phe Leu Ala Asp ValGlu Gly Leu Ser 755 760 765 Val Leu Arg Ser Phe Arg Leu Leu Arg Val PheLys Leu Ala Lys Ser 770 775 780 Trp Pro Thr Leu Asn Met Leu Ile Lys IleIle Gly Asn Ser Val Gly 785 790 795 800 Ala Leu Gly Asn Leu Thr Leu ValLeu Ala Ile Ile Val Phe Ile Phe 805 810 815 Ala Val Val Gly Met Gln LeuPhe Gly Lys Ser Tyr Lys Glu Cys Val 820 825 830 Cys Lys Ile Asn Asp CysLeu Pro Arg Trp His Met Asn Asp Phe Phe 835 840 845 His Ser Phe Leu IleVal Phe Arg Val Leu Cys Gly Glu Trp Ile Glu 850 855 860 Thr Met Trp AspCys Met Glu Val Ala Gly Gln Met Cys Leu Ile Val 865 870 875 880 Tyr MetMet Val Met Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe 885 890 895 LeuAla Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu Thr Ala Ile 900 905 910Glu Glu Asp Asp Ala Asn Asn Leu Gln Ile Ala Val Arg Ile Lys Gly 915 920925 Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu Phe Ile Leu Lys Phe Ser 930935 940 Lys Lys Pro Lys Ser Asp Asn Lys Lys Glu Asn Tyr Ile Ser Asn Thr945 950 955 960 Leu Ala Glu Met Ser Lys His Asn Phe Leu Lys Glu Lys AspIle Ser 965 970 975 Gly Gly Ser Ser Asp Lys Met Asp Gln Ser Phe Ile HisAsn Pro Ser 980 985 990 Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu SerAsp Leu Glu Met 995 1000 1005 Asn Glu Glu Leu Ser Ser Asp Ser Asp SerTyr Ser Lys Asn Arg Ser 1010 1015 1020 Ser Ser Ser Glu Cys Ser Thr ValAsp Asn Pro Leu Pro Gly Glu Gly 1025 1030 1035 1040 Glu Glu Ala Glu AlaGlu Pro Asn Asp Glu Pro Glu Ala Cys Phe Thr 1045 1050 1055 Asp Gly CysVal Arg Arg Phe Cys Cys Gln Val Asn Ser Gly Lys Gly 1060 1065 1070 LysTrp Trp Ile Arg Lys Thr Cys Tyr Ile Val Glu His Ser Trp Phe 1075 10801085 Glu Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala1090 1095 1100 Phe Glu Asp Ile Tyr Ile Glu Lys Lys Thr Ile Lys Ile IleLeu Glu 1105 1110 1115 1120 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe IleLeu Glu Met Leu Leu 1125 1130 1135 Lys Trp Ala Tyr Gly Tyr Lys Thr TyrPhe Thr Asn Ala Trp Cys Trp 1140 1145 1150 Leu Asp Phe Leu Ile Val AspVal Ser Leu Val Thr Leu Val Ala Asn 1155 1160 1165 Thr Leu Gly Tyr SerAsp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu 1170 1175 1180 Arg Ala LeuArg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg 1185 1190 1195 1200Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn Val 12051210 1215 Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met GlyVal 1220 1225 1230 Asn Leu Phe Ala Gly Lys Phe Tyr Glu Cys Asn Thr ThrAsp Gly Ser 1235 1240 1245 Arg Phe Pro Ser Gln Val Asn Arg Ser Glu CysPhe Ala Leu Met Asn 1250 1255 1260 Val Ser Asn Val Arg Trp Lys Asn LeuLys Val Asn Phe Asp Asn Val 1265 1270 1275 1280 Gly Leu Gly Tyr Leu SerLeu Leu Gln Val Ala Thr Phe Lys Gly Trp 1285 1290 1295 Ile Met Tyr AlaAla Val Asp Ser Val Asn Val Gln Pro Lys Tyr Glu 1300 1305 1310 Tyr SerLeu Tyr Met Tyr Ile Tyr Phe Val Phe Ile Ile Phe Gly Ser 1315 1320 1325Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn 13301335 1340 Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met Thr GluGlu 1345 1350 1355 1360 Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu GlySer Lys Lys Pro 1365 1370 1375 Gln Lys Pro Ile Pro Arg Pro Gly Asn LysGln Gly Cys Ile Phe Asp 1380 1385 1390 Leu Thr Asn Gln Ala Phe Asp IleIle Met Val Leu Ile Cys Leu Asn 1395 1400 1405 Met Val Thr Met Met ValGlu Lys Glu Gly Gln Met Val Leu Trp Ile 1410 1415 1420 Asn Val Phe IleIle Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile 1425 1430 1435 1440 SerLeu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn Ile Phe Val Val 1445 14501455 Val Ile Ser Ile Val Gly Met Phe Leu Ala Ile Glu Tyr Phe Val Ser1460 1465 1470 Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly ArgIle Leu 1475 1480 1485 Arg Leu Lys Gly Ala Lys Gly Ile Arg Thr Leu LeuPhe Ala Leu Met 1490 1495 1500 Met Ser Leu Pro Ala Leu Phe Asn Ile GlyLeu Leu Leu Phe Leu Val 1505 1510 1515 1520 Met Phe Ile Tyr Ala Ile PheGly Met Ser Asn Phe Ala Tyr Val Lys 1525 1530 1535 Lys Glu Gly Ile AsnAsp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser 1540 1545 1550 Met Ile CysLeu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu 1555 1560 1565 LeuAla Pro Ile Leu Asn Ser Pro Pro Asp Cys Asp Pro Lys Lys Val 1570 15751580 His Pro Gly Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly1585 1590 1595 1600 Ile Phe Tyr Phe Val Ser Tyr Ile Ile Ile Ser Phe LeuVal Val Val 1605 1610 1615 Asn Met Tyr Ile Ala Val Ile Leu Glu Asn PheSer Val Ala Thr Glu 1620 1625 1630 Glu Ser Thr Glu Pro Leu Ser Glu AspAsp Phe Glu Met Phe Tyr Glu 1635 1640 1645 Val Trp Glu Lys Phe Asp ProAsp Ala Thr Gln Phe Ile Glu Phe Lys 1650 1655 1660 Leu Ser Asp Phe AlaAla Ala Leu Asp Pro Pro Leu Leu Ile Ala Lys 1665 1670 1675 1680 Pro AsnLys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly 1685 1690 1695Asp Arg Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val 17001705 1710 Leu Gly Glu Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu GluArg 1715 1720 1725 Phe Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu ProIle Thr Thr 1730 1735 1740 Thr Leu Lys Arg Lys Gln Glu Val Ser Ala ThrIle Gln Arg Ala Tyr 1745 1750 1755 1760 Arg Arg Tyr Arg Leu Arg Gln ValLys Asn Ile Ser Ser Ile Tyr Ile 1765 1770 1775 Lys Asp Gly Asp Arg AspAsp Asp Leu Asn Lys Asp Phe Asp Asn Val 1780 1785 1790 Asn Glu Asn SerSer Pro Glu Lys Thr Asp Thr Ser Thr Ser Pro Pro 1795 1800 1805 Ser TyrAsp Ser Val Thr Lys Pro Asp Glu Lys Tyr Glu Asp Thr Glu 1810 1815 1820Lys Glu Asp Lys Lys Asp Ser Lys Glu Ser Lys 1825 1830 1835 1969 aminoacids amino acid Not Relevant linear protein 16 Met Ala Met Leu Pro ProPro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu AlaLeu Ile Glu Gln Arg Ile Ala Glu Arg Lys Ser 20 25 30 Lys Glu Pro Lys GluGlu Lys Lys Asp Asp Asp Glu Glu Ala Pro Lys 35 40 45 Pro Ser Ser Asp LeuGlu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp Ile Pro Pro GlyMet Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 Tyr Tyr Ala AspLys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Ala 85 90 95 Ile Phe Arg PheAsn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110 Ser Pro LeuArg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120 125 Ser MetLeu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130 135 140 MetAsn Asn Pro Pro Asp Trp Thr Lys Asn Val Gly Tyr Thr Phe Thr 145 150 155160 Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys Ile Leu Ala Arg Gly Phe 165170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val Asn LeuGly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala LeuLys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly AlaLeu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp Val Met Ile LeuThr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile Gly Leu Gln LeuPhe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg Asn Ser Leu GluAsn Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr Leu Glu Ser GluGlu Asp Phe Arg Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu Gly Ser Lys AspAla Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser Gly Gln Cys ProGlu Gly Tyr Thr Cys Val Lys Ile Gly Arg Asn 325 330 335 Pro Asp Tyr GlyTyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350 Ala Leu PheArg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360 365 Gln ThrLeu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370 375 380 ValIle Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val 385 390 395400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala 405410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp Arg Leu Lys Lys420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala Ala Ala Glu TyrThr 435 440 445 Ser Ile Arg Arg Ser Arg Ile Met Gly Leu Ser Glu Ser SerSer Glu 450 455 460 Thr Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu Arg ArgAsn Arg Arg 465 470 475 480 Lys Lys Lys Asn Gln Lys Lys Leu Ser Ser GlyGlu Glu Lys Gly Asp 485 490 495 Ala Glu Lys Leu Ser Lys Ser Glu Ser GluAsp Ser Ile Arg Arg Lys 500 505 510 Ser Phe His Leu Gly Val Glu Gly HisArg Arg Ala His Glu Lys Arg 515 520 525 Leu Ser Thr Pro Asn Gln Ser ProLeu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala Arg Arg Ser Ser ArgThr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 Gly Arg Asp Xaa GlySer Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575 Ile Phe Gly AspAsn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585 590 Arg Pro XaaGlu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595 600 605 Pro ProMet Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys 610 615 620 AsnGly Val Val Ser Leu Val Asp Gly Xaa Ser Ala Leu Met Leu Pro 625 630 635640 Asn Gly Gln Leu Leu Pro Glu Gly Thr Thr Asn Gln Ile His Lys Lys 645650 655 Arg Arg Cys Ser Ser Tyr Leu Leu Ser Glu Asp Met Leu Asn Asp Pro660 665 670 Asn Leu Arg Gln Arg Ala Met Ser Arg Ala Ser Ile Leu Thr AsnThr 675 680 685 Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro Pro TrpTrp Tyr 690 695 700 Arg Phe Ala His Lys Phe Leu Ile Trp Asn Cys Ser ProTyr Trp Ile 705 710 715 720 Lys Phe Lys Lys Cys Ile Tyr Phe Ile Val MetAsp Pro Phe Val Asp 725 730 735 Leu Ala Ile Thr Ile Cys Ile Val Leu AsnThr Leu Phe Met Ala Met 740 745 750 Glu His His Pro Met Thr Glu Glu PheLys Asn Val Leu Ala Ile Gly 755 760 765 Asn Leu Val Phe Thr Gly Ile PheAla Ala Glu Met Val Leu Lys Leu 770 775 780 Ile Ala Met Asp Pro Tyr GluTyr Phe Gln Val Gly Trp Asn Ile Phe 785 790 795 800 Asp Ser Leu Ile ValThr Leu Ser Leu Val Glu Leu Phe Leu Ala Asp 805 810 815 Val Glu Gly LeuSer Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe 820 825 830 Lys Leu AlaLys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile 835 840 845 Gly AsnSer Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile 850 855 860 IleVal Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser 865 870 875880 Tyr Lys Glu Cys Val Cys Lys Ile Asn Asp Asp Cys Thr Leu Pro Arg 885890 895 Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg Val900 905 910 Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu ValAla 915 920 925 Gly Gln Ala Met Cys Leu Ile Val Tyr Met Met Val Met ValIle Gly 930 935 940 Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu LeuSer Ser Phe 945 950 955 960 Ser Ser Asp Asn Leu Thr Ala Ile Glu Glu AspPro Asp Ala Asn Asn 965 970 975 Leu Gln Ile Ala Val Thr Arg Ile Lys LysGly Ile Asn Tyr Val Lys 980 985 990 Gln Thr Leu Arg Glu Phe Ile Leu LysAla Phe Ser Lys Lys Pro Lys 995 1000 1005 Ile Ser Arg Glu Ile Arg GlnAla Glu Asp Leu Asn Thr Lys Lys Glu 1010 1015 1020 Asn Tyr Ile Ser AsnMet Thr Leu Ala Glu Met Ser Lys Gly His Asn 1025 1030 1035 1040 Phe LeuLys Glu Lys Asp Lys Ile Ser Gly Phe Gly Ser Ser Xaa Asp 1045 1050 1055Lys His Leu Met Glu Asp Ser Asp Gly Gln Ser Phe Ile His Asn Pro 10601065 1070 Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp LeuGlu 1075 1080 1085 Met Asn Glu Glu Leu Ser Ser Asp Ser Asp Ser Tyr SerLys Asn Arg 1090 1095 1100 Ser Ser Ser Ser Glu Cys Ser Thr Val Asp AsnPro Leu Pro Gly Glu 1105 1110 1115 1120 Gly Glu Glu Ala Glu Ala Glu ProAsn Asp Glu Pro Glu Ala Cys Phe 1125 1130 1135 Thr Asp Gly Cys Val ArgArg Phe Ser Cys Cys Gln Val Asn Ile Glu 1140 1145 1150 Ser Gly Lys GlyLys Ile Trp Trp Asn Ile Arg Lys Thr Cys Tyr Lys 1155 1160 1165 Ile ValGlu His Ser Trp Phe Glu Ser Phe Ile Val Leu Met Ile Leu 1170 1175 1180Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile Glu Arg Lys 11851190 1195 1200 Lys Thr Ile Lys Ile Ile Leu Glu Tyr Ala Asp Lys Ile PheThr Tyr 1205 1210 1215 Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Ile AlaTyr Gly Tyr Lys 1220 1225 1230 Thr Tyr Phe Thr Asn Ala Trp Cys Trp LeuAsp Phe Leu Ile Val Asp 1235 1240 1245 Val Ser Leu Val Thr Leu Val AlaAsn Thr Leu Gly Tyr Ser Asp Leu 1250 1255 1260 Gly Pro Ile Lys Ser LeuArg Thr Leu Arg Ala Leu Arg Pro Leu Arg 1265 1270 1275 1280 Ala Leu SerArg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu Ile 1285 1290 1295 GlyAla Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile Phe 1300 13051310 Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys Phe1315 1320 1325 Tyr Glu Cys Ile Asn Thr Thr Asp Gly Ser Arg Phe Pro AlaSer Gln 1330 1335 1340 Val Pro Asn Arg Ser Glu Cys Phe Ala Leu Met AsnVal Ser Gln Asn 1345 1350 1355 1360 Val Arg Trp Lys Asn Leu Lys Val AsnPhe Asp Asn Val Gly Leu Gly 1365 1370 1375 Tyr Leu Ser Leu Leu Gln ValAla Thr Phe Lys Gly Trp Thr Ile Ile 1380 1385 1390 Met Tyr Ala Ala ValAsp Ser Val Asn Val Asp Lys Gln Pro Lys Tyr 1395 1400 1405 Glu Tyr SerLeu Tyr Met Tyr Ile Tyr Phe Val Val Phe Ile Ile Phe 1410 1415 1420 GlySer Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn 1425 14301435 1440 Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe MetThr 1445 1450 1455 Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys LeuGly Ser Lys 1460 1465 1470 Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly AsnLys Ile Gln Gly Cys 1475 1480 1485 Ile Phe Asp Leu Val Thr Asn Gln AlaPhe Asp Ile Ser Ile Met Val 1490 1495 1500 Leu Ile Cys Leu Asn Met ValThr Met Met Val Glu Lys Glu Gly Gln 1505 1510 1515 1520 Ser Gln His MetThr Glu Val Leu Tyr Trp Ile Asn Val Val Phe Ile 1525 1530 1535 Ile LeuPhe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser Leu Arg His 1540 1545 1550Tyr Tyr Phe Thr Val Gly Trp Asn Ile Phe Asp Phe Val Val Val Ile 15551560 1565 Ile Ser Ile Val Gly Met Phe Leu Ala Asp Leu Ile Glu Thr TyrPhe 1570 1575 1580 Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala ArgIle Gly Arg 1585 1590 1595 1600 Ile Leu Arg Leu Val Lys Gly Ala Lys GlyIle Arg Thr Leu Leu Phe 1605 1610 1615 Ala Leu Met Met Ser Leu Pro AlaLeu Phe Asn Ile Gly Leu Leu Leu 1620 1625 1630 Phe Leu Val Met Phe IleTyr Ala Ile Phe Gly Met Ser Asn Phe Ala 1635 1640 1645 Tyr Val Lys LysGlu Asp Gly Ile Asn Asp Met Phe Asn Phe Glu Thr 1650 1655 1660 Phe GlyAsn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly 1665 1670 16751680 Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Lys Pro Pro Asp Cys1685 1690 1695 Asp Pro Lys Lys Val His Pro Gly Ser Ser Val Glu Gly AspCys Gly 1700 1705 1710 Asn Pro Ser Val Gly Ile Phe Tyr Phe Val Ser TyrIle Ile Ile Ser 1715 1720 1725 Phe Leu Val Val Val Asn Met Tyr Ile AlaVal Ile Leu Glu Asn Phe 1730 1735 1740 Ser Val Ala Thr Glu Glu Ser ThrGlu Pro Leu Ser Glu Asp Asp Phe 1745 1750 1755 1760 Glu Met Phe Tyr GluVal Trp Glu Lys Phe Asp Pro Asp Ala Thr Gln 1765 1770 1775 Phe Ile GluPhe Ser Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro 1780 1785 1790 ProLeu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile Ala Met Asp 1795 18001805 Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp Ile Leu Phe1810 1815 1820 Ala Phe Thr Lys Arg Val Leu Gly Glu Ser Gly Glu Met AspSer Leu 1825 1830 1835 1840 Arg Ser Gln Met Glu Glu Arg Phe Met Ser AlaAsn Pro Ser Lys Val 1845 1850 1855 Ser Tyr Glu Pro Ile Thr Thr Thr LeuLys Arg Lys Gln Glu Xaa Val 1860 1865 1870 Ser Ala Thr Val Ile Gln ArgAla Tyr Arg Arg Tyr Arg Leu Arg Gln 1875 1880 1885 Asn Val Lys Asn IleSer Ser Ile Tyr Ile Lys Asp Gly Asp Arg Asp 1890 1895 1900 Asp Asp LeuLeu Asn Lys Glu Asp Met Ala Phe Asp Asn Val Asn Glu 1905 1910 1915 1920Asn Ser Ser Pro Glu Lys Thr Asp Ala Thr Ser Ser Thr Thr Ser Pro 19251930 1935 Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Lys Glu Lys Tyr GluXaa 1940 1945 1950 Asp Gln Thr Glu Lys Glu Asp Lys Gly Lys Asp Ser LysGlu Ser Lys 1955 1960 1965 Lys 21 base pairs nucleic acid single linearcDNA 17 TTTGTGCCCC ACAGACCCCA G 21 26 base pairs nucleic acid singlelinear cDNA 18 ACACAAATTC TTGATCTGGA ATTGCT 26 23 base pairs nucleicacid single linear cDNA 19 CAACCTCAGA CAGAGAGCAA TGA 23

1-16. (Cancelled)
 17. A bioassay for assessing a candidate modulatingagent of a PNS SCP, comprising: (A) contacting a candidate agent with acell line expressing in the cell membrane of said cell a PNS SCP; and(B) evaluating the modulation of the SC biological activity of said cellmediated by said contacting of said candidate agent.
 18. A methodaccording to claim 17, wherein said cell line is selected from PC12cells or a recombinant form thereof comprising an isolated nucleic acidmolecule, wherein the isolated nucleic acid molecule encodes apolypeptide monomer of a peripheral nervous system specific (PNS) sodiumchannel the monomer: (i) having an amino acid sequence that has at least87% identity to the full amino acid sequence disclosed in SEQ ID NO:10and (ii) forming a PNS type I voltage gated sodium channel sodium ionswhen the monomer is expressed in Xenopus oocytes.
 19. A PNS SCPmodulating agent, identified by a method according to claim
 17. 20. APNS SCP modulating agent according to claim 19, wherein said agent is amethyl-phenylhalophenyl-substituted piperizine compound.
 21. A PNS SCPmodulating agent according to claim 20, wherein said piperizine compoundis lidoflazine (Merck Index Monograph 5311) or a derivative thereof.22-39. (Cancelled)